Responder spleen cells of [B10.A(5R) x B10.A(2R)]F1 hybrid mice cultured in vitro with irradiated parental spleen cells, generate a cytotoxic T cell response which maps to the H-2Dd and H-2(K-I)b genes of the B10.A(5R) parent. Specific reactivity of (5R x 2R) cells to B10.A(5R) parental (P) stimulators and not to F1 hybrid or B10.A(2R) stimulators was also demonstrated as an F1 anti-P proliferative response. Generation of a (5R x 2R) anti-5R response has also been noted during 2 degrees in vitro anti-viral cytotoxic responses where (5R x 2R) cells from either ectromelia or Sendai virus primed mice have been stimulated in vitro with infected 5R cells, but not when stimulated with infected 2R or (5R x 2R) cells. The generation of these responses is dependent on the use of parental stimulators for F1 hybrid cells during in vitro culture. All data is consistent with the hypothesis that (5R x 2R) F1 cells are not tolerant of 5R cells, and of the 4-fold higher level of Class I H-2Kb/Dd antigens which they express.