Cultured human keratinocytes are used for skin grafts, but their success is limited by late graft loss. Development of antibody to fetal bovine serum (FBS) protein used in culture media for in vitro keratinocyte growth has been identified. The persistence of FBS antigen in skin grafts is important in the induction of the immune response and the susceptibility of the keratinocytes to immune-mediated injury. The magnitude and longevity of FBS protein persistence on human keratinocytes was studied. Secondary passage human keratinocytes were grown in media supplemented with 5% FBS. The media was changed to one supplemented with pooled human AB serum, and the amount of FBS protein incorporated in the tissue was measured over the following 8 days by an ELISA reaction directed against FBS antigen. Incorporated FBS antigen decreased for the first 3 days to 31% of maximum. There was no further significant decrease for 5 days. Keratinocytes grown in alternative serum supplements (NuSerum [Collaborative Research Inc., Bedford, Mass.] and Serum Plus [Hazelton Research Products Inc., Lenexa, Kan.]), which contain reduced amounts of FBS, offered no significant reduction in FBS protein incorporation. This duration of antigen persistence would make human keratinocytes susceptible to cell destruction by immune response to FBS and may contribute to delayed loss of human keratinocyte grafts.