Maria Sharapova was forced out of her second-round match at the Italian Open through injury, having earlier been told she would not be handed a wild card for the French Open.
The 30-year-old former world number one had competed at three events since returning to competitive tennis following her 15-month doping suspension, reaching the semi-final of the Stuttgart Open, but had not been able to accrue enough ranking points to secure another return to Roland Garros.
Sharapova, a two-time French Open champion, requested one of the wild card slots, but on Tuesday afternoon French Tennis Federation president Bernard Giudicelli confirmed via a live Facebook broadcast it would not be granted by the tournament organisers.
Giudicelli is understood to have spoken to Sharapova - who tested positive for meldonium at the 2016 Australian Open and then saw her original two-year ban reduced on appeal - ahead of the decision being announced, with the Russian taking to the court in Rome against Croatian Mirjana Lucic-Baroni not long after.
After a shaky start when losing her opening service game and subsequently the first set 6-4, Sharapova looked on course for a recovery as she levelled the match before then breaking the number 16 early in the decider.
However, Sharapova, currently ranked 211, had gone off for some treatment after the opening game of the third set, returning with her upper left leg strapped to hold serve, but then lacked the mobility to force home any of the four break chances in game three and so opted to retire.
Lucic-Baroni will go on to face either top seed Angelique Kerber, the current world number one, or Anett Kontaveit in the third round.
The future for Sharapova, meanwhile, remains uncertain.
After an early exit from the Italian Open, the Russian can now no longer reach the main draw at Wimbledon from ranking, although she could still come through qualifying or yet be handed a wild card by the All England Lawn and Tennis Club on June 20.
Sharapova, who won Wimbledon in 2004 as an unheralded teenager, is reported to have been offered a place at the WTA Aegon Classic Birmingham in the run-up to SW19, where it remains to be seen if she will be fit enough to compete anyway should her leg injury prove serious.
In the meantime, though, Sharapova's clay-court campaign will not now take in the French Open, where she lifted the title in 2012 and 2014.
FTF president Giudicelli said during the live Facebook broadcast: "I just wanted to tell you that I decided not to give to Maria Sharapova a wild card, a wild card she asked me for.
"Nobody can deprive her of her two titles here in Roland Garros, but these two titles she had conquered them according to the rules and behold nothing to anyone."
Giudicelli continued: "Today this (doping) suspension is over and she can take her path towards the new success, but if there can be a wild card for return from injuries then there cannot be a wild card for return from doping.
"So it is up to her, day after day, tournament after tournament, to find alone the strength to conquer major titles without being held to anybody.
"I'm very sorry for Maria, very sorry for her fans. They might be very disappointed and she might be very disappointed, but it is my responsibility and my mission to protect the game and protect high standards of the game played without any 'doping' on the result, so that is our decision."
Giudicelli noted there were also strong calls for Sharapova to be offered a place at Roland Garros, but stressed it was "inconceivable to take a decision" against the anti-doping efforts of tournaments and governing bodies.
He added: "Of course I felt some pressure. We did not want to treat Maria Sharapova differently.... Maria won twice here, but we have a huge responsibility.
"I know the media dimension Maria has, I know the expectation fans and broadcasters have, but it didn't seem possible for me to go above the strong commitment and the respect for the anti-doping code."
Motivated more than usual, Eugenie Bouchard used her game to send a message to Maria Sharapova by defeating the Russian 7-5, 2-6, 6-4 in a thrilling second-round match at the Madrid Open on Monday.
Eugenie Bouchard produced a stunning performance to beat Maria Sharapova at the Mutua Madrid Open on Monday night, with the frosty handshake exchanged between the two players after their epic contest evidence of the animosity that hung over their grudge match.