The experiments reported here were designed to investigate the role of major histocompatibility antigens alone in primary allogeneic responses in vitro. Mixed lymphocyte cultures were established using small numbers of responder spleen cells and semiallogeneic or fully allogeneic stimulator spleen cells. The cytotoxicity generated in individual cultures was then detected by a sensitive 51Cr-release assay and the frequencies of responding cytotoxic T lymphocyte precursors (CTL-P) determined by limit dilution analysis. To investigate the responses of spleen cells to major histocompatibility (H-2) antigens alone, strains of BALB/c mice congenic at the H-2 locus were used as responder and stimulator cells. The frequencies of CTL-P responding in such congenic combinations were compared with those obtained using mouse strain combinations differing at minor histocompatibility (minor-H) loci in addition to H-2. A similar fraction of spleen cells responded regardless of whether the stimulator cells differed at the H-2 locus alone or at multiple minor-H loci in addition to H-2. It was concluded that minor-H antigenic differences do not contribute significantly to allogeneic responses.