Although cultured keratinocyte allografts are initially accepted, grafted donor cells are gradually replaced by recipient elements. The precise mechanisms underlying this process are not clear. Keratinocyte stem cells (KSCs) were isolated and purified from neonatal C57BL/6 (H-2(b)) and BALB/c (H-2(d)) mice. Cultured KSCs were negative for IA/IE, indicating the absence of any contamination with Langerhans cells. Immunostaining showed that over a 7-day culture period, KSCs expressed increasing amounts of CD80, while CD86 could not be detected. RT-PCR results were consistent with these data. Mixed lymphocyte reaction assays showed that KSCs cultured in 10% serum for 7 days stimulated allogeneic rather than syngeneic T-cell proliferation. This study demonstrates that the costimulatory molecule CD80, but not CD86, may be expressed on cultured KSCs. The data indicate KSCs could act as antigen-presenting cells and thus provide an alternative explanation for the ultimate rejection of allogeneic keratinocytes.