The timing of the attachment of fatty acids to the hemagglutinin (HA) of influenza A virus was studied. Treatment of virus infected cells with brefeldin A (BFA), a drug which blocks intracellular transport along the exocytic pathway at a pre-Golgi site, does not prevent palmitoylation of HA. The relationship of HA-palmitoylation to the oligomerisation and to the proteolytical cleavage of the protein revealed that the uncleaved trimer of HA is the substrate for the acylating enzyme in virus infected cells. The results are discussed with regard to the intracellular site of palmitoylation.