Valentino Rossi was Friday cleared to race in the European MotoGP after recovering from coronavirus but Yamaha's joy at seeing the return of their seven-time world champion was tempered when title-chasing Maverick Vinales was ordered to start Sunday's race from the pit lane.
Rossi flew to Valencia from Italy on Thursday evening after a first negative Covid-19 test.
"This morning, Rossi took a second PCR test, as per FIM (International Motorcycling Federation) regulations, and this afternoon it came back negative," said Yamaha in a statement.
"The two consecutive negative test results allow Rossi to reunite with the team and take part in this weekend's Gran Premio de Europa."
The veteran Italian did not participate in Friday's opening two practice sessions in Valencia.
American stand-in Garrett Gerloff managed 19th place.
Rossi, 41, missed back-to-back races at MotorLand Aragon last month after testing positive for Covid-19.
He stands 15th in the overall standings.
Meanwhile, Vinales, who is just 19 points off the championship lead, will start Sunday's race from the pit lane, five seconds after the green light is shown at the exit.
Vinales was penalised after exceding the permitted number of engines.
Yamaha also revealed that they will not appeal their 50-point deduction slapped on them by the FIM "failing to respect the protocol required for technical changes".
On the track, Australian Jack Miller produced the best time in Friday's practice while championship rivals Joan Mir and Fabio Quartararo cracked the top 10 despite struggling in a wet opening session.
After dominating on a slick track in the morning practice, Ducati rider Miller set a fastest lap of 1min 32.528sec as the Ricardo Tormo circuit dried in the afternoon session.
Aleix Espargaro was a surprise second ahead of Franco Morbidelli, Quartararo's Yamaha-SRT team-mate and the Teruel Grand Prix from winner two weeks ago.
Quartararo bounced back to finish ninth in the second session having come 21st and last earlier in the day.
Mir, who leads the MotoGP standings by 14 points, was 10th with both riders over 0.8sec behind Miller.
South African Brad Binder, seventh on Friday, will serve a long lap penalty in Sunday's race as punishment for the KTM rider's collision with Miller in the previous grand prix.
- 'More pressure' -
Quartararo, who faded badly after starting two of the past three races on pole, showed his frustration by banging his fist on his fuel tank after sliding off the track in soggy morning conditions.
But the French rider, who has won three races this season, moved up the timesheets as the weather improved for the second session.
Mir had initially fared little better than Quartararo in the first run out when he finished 1.6 seconds adrift of Miller in 17th place.
The Valencia MotoGP will be held on the same circuit next weekend before the season finale takes place at Portimao in Portugal on November 22.
Mir has admitted the pressure is beginning to tell as he closes in on the possibility of becoming world champion without winning a race.
With three rounds left, there are still 75 points available.
Only 32 points separate the top six after a roller-coaster season truncated by the coronavirus and thrown wide open by the absence of six-time world champion Marc Marquez.
The Spaniard hasn't raced since breaking his arm in the season-opener in Jerez.
"For sure, there is the feeling of a little bit more pressure because the championship is getting older, so every one of us feels like it's going to finish soon," said Suzuki rider Mir who has a 14-point lead over Quartararo.
He may not have been a race winner, but Mir is the picture of consistency in 2020 with three second places and three thirds from 11 races.
French star Quartararo may have three wins to his name in 2020 but the Yamaha satellite rider endured miserable back-to-back races in Aragon in October.
He was a lowly 18th and then eighth in races won respectively by Alex Rins on a Suzuki and Morbidelli.
At the same time, Mir picked up a couple of third-place finishes in a perfect summary of his year so far.
Vinales, whose lone win in 2020 came at Emilia Romagna, is five points behind Quartararo.
He was fourth and seventh at Aragon, losing ground on Mir but closing in on his French rival.