A normal female donor (H9) is described, whose cells generate strong cytotoxicity against a human minor histocompatibility antigen in vitro. These cytotoxic T lymphocytes are generated after secondary restimulation with cells from an HLA-A, -B, -C, and -DR-matched donor and are HLA restricted (HLA-B7). No other donor could be identified whose cells responded to this antigen. The two children of donor H9 are virtually HLA identical to her and one of the children expresses the relevant minor histocompatibility antigen. These data suggest that priming in vivo during pregnancy has facilitated cytotoxic T cell response to human minor histocompatibility antigens in vitro.