South Africa's double World Cup-winning wing Cheslin Kolbe left the Springbok training camp on Monday for tests on a "knee niggle", SA Rugby announced in a statement.
The 30-year-old was injured while playing for his Japanese club Tokyo Sungoliath.
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Kolbe, who was named among 35 players for the Boks' first training camp of the year, joined his team-mates on Sunday evening but was released on Monday "to examine the extent of niggle which he picked up during his regular Japanese club season".
He has been replaced in the camp by uncapped Bulls back-row forward Celimpilo Gumede.
"Celimpilo has been playing impressive rugby this season and we are excited to see how he integrates in the squad and what he has to offer at training," said Springbok head coach Rassie Erasmus.
It continues the injury issues with South Africa captain Siya Kolisi one of 12 further players from the matchday 23 who won the Rugby World Cup last year who were not be considered for a one-off Test against Wales at Twickenham on June 22 when the squad was selected on Sunday.
"Players based in Europe, the UK and Ireland will not be considered for the Boks’ first Test of the season against Wales at Twickenham," SA Rugby said in a statement.
Kolisi moved to Paris after the World Cup triumph to join Racing92 who on Saturday qualified for next week's Top 14 play-offs, with the semi-finals on the same weekend as the Wales Test.
The match is taking place outside the international window so teams beyond South Africa do not have to release players.
With the Japanese season completed, however, eight South Africans based there were selected by head coach Rassie Erasmus on Sunday.
Veteran lock Eben Etzebeth is considered a strong candidate to skipper the world champion Springboks in a match that serves as a warm-up for two home Tests against Ireland during July.
Erasmus, who played different but key coaching roles in the 2019 and 2023 World Cup triumphs, named 18 forwards and 17 backs for a training camp in Pretoria, which begins on Monday.
He included fly-half Handre Pollard and No. 8 Jasper Wiese, but neither will be considered for Twickenham.
Wiese, the likely successor to retired Duane Vermeulen in the back row, was hit with a six-Test ban after being sent off in an English Premiership match, and it starts against Wales.
Pollard was a key figure in the 2023 World Cup triumph. Initially ruled out of the tournament by injury, he was called up when hooker Malcolm Marx was sidelined.
The playmaker did not miss a single kick at goal in tense, one-point knockout victories over hosts France, England and New Zealand.
With Pollard out of the reckoning for Twickenham, Manie Libbok and uncapped pair Siya Masuku and Jordan Hendrikse will be the fly-half contenders.
Hendrikse and Masuku are among 11 uncapped players in the squad. Another is hooker Andre-Hugo Venter, a son of Springbok legend Andre Venter.
South Africa face Wales at Twickenham before going into a busy schedule that includes two Tests against Ireland and one against Portugal before they embark on the Rugby Championship, which involves six matches against Australia, arch-rivals New Zealand and Argentina in August and September.
The four-time world champions then take on Scotland, England and Wales during a November tour of Europe.
Forwards
Joseph Dweba, Ben-Jason Dixon, Neethling Fouche, Frans Malherbe, Salmaan Moerat, Evan Roos, Andre-Hugo Venter (all Stormers), Phepsi Buthelezi, Eben Etzebeth, Vincent Koch, Bongi Mbonambi, Ntuthuko Mchunu, Ox Nche (all Sharks), Malcolm Marx (Kubota Spears/JPN), Franco Mostert (Honda Heat/JPN), Kwagga Smith (Blu Revs/JPN), Pieter-Steph du Toit (Toyota Verblitz/JPN), Jasper Wiese (Leicester/ENG)
Backs
Aphelele Fassi, Ethan Hooker, Makazole Mapimpi, Siya Masuku, Grant Williams (all Sharks), Morne van den Berg, Jordan Hendrikse, Quan Horn, Edwill van der Merwe (all Lions), Sacha Feinberg-Mngomezulu, Manie Libbok (both Stormers), Faf de Klerk, Jesse Kriel (both Canon Eagles/JPN), Damian de Allende (Panasonic Wild Knights/JPN), Andre Esterhuizen (Harlequins/ENG), Cheslin Kolbe (Suntory Sungoliath/JPN), Handre Pollard (Leicester/ENG)
