Striker Diafra Sakho and West Ham face a Fifa inquiry over his withdrawal from Senegal's Africa Cup of Nations squad.
Football's governing body says it has opened disciplinary proceedings for "a potential violation of regulations".
Sakho, 25, withdrew with a back injury, but later scored for the Hammers in their 1-0 FA Cup win at Bristol City.
Fifa rules say a player cannot appear for a club if he was meant to be on international duty, but West Ham "vigorously" deny any wrongdoing.
A Fifa spokesman said: "We can confirm that disciplinary proceedings were initiated against the player Diafra Sakho and the club West Ham United for a potential violation of the Fifa regulations on the status and transfer of players."
He declined to give a likely time frame for the case, or potential penalties, should it find that the rules were breached.
A West Ham statement said: "Diafra Sakho and West Ham United vigorously deny that they have acted in breach of Fifa regulations. They regard the proceedings as clearly unfounded and are requesting their dismissal."
Sakho was not in West Ham's squad for Saturday's 2-0 Premier League defeat at Liverpool, with manager Sam Allardyce saying he had been advised not to play him until the dispute was resolved.
On Thursday, Allardyce said he was confident the striker - who has scored eight times in 12 Premier League games since signing from FC Metz in August - would not face a ban.
Senegal boss Alain Giresse, who was told Sakho had withdrawn from the Africa Cup of Nations as he could not fly because of a back injury, claimed West Ham had "set a dangerous precedent" by playing him.
Bristol City are not commenting on reports they could seek compensation of £250,000 if Fifa rules that Sakho should not have played in the fourth-round FA Cup tie.
West Ham visit West Brom in the fifth round on 15 February.