Cytotoxic T lymphocyte (CTL) responses of splenic T cells from C57BL/6 B6) mice and mutant H-2Kbm1 (bm1) mice to haptenic (trinitrophenyl [TNP] ) and herpes simplex virus (HSV) determinants in the context of an allogenic (wild-type or mutant) H-2Kb molecule were analyzed in a modified limiting dilution system. In the B6-anti-bm1TNP mixed leukocyte reaction (MLR), estimated frequencies for precursors of CTL clones that lysed bm1TNP targets ranged from 1/120 to 1/400; in the bm1-anti-B6TNP MLR, estimated frequencies of precursors of CTL clones that lysed B6TNP targets ranged from 1/500 to 1/1,300. Estimated frequencies for precursors of CTL clones that lysed the respective unmodified and TNP-modified allogeneic targets were two- to three-fold lower. Lytic specificity patterns determined by split-well analysis showed that at least 20-30% of the generated CTL populations (selected for a high probability of clonality) in both MLR displayed allorestricted lysis of TNP-modified concanavalin A blast targets. In the B6-anti-bm1HSV MLR, estimated frequencies for precursors of CTL clones that lysed bm1HSV targets ranged from 1/70 to 1/300; in the bm1-anti-B6HSV MLR, estimated frequencies for precursors of CTL clones that lysed B6HSV targets ranged from 1/300 to 1/1,200. Again, estimated frequencies for precursors of CTL clones that lysed the respective noninfected and virus-infected allogeneic targets were two- to fourfold lower. Of the CTL populations selected for a high probability of clonality at least 30-60% displayed allorestricted lysis of virus-infected lipopolysaccharide blast targets in both MLR. It is concluded that a large fraction of clonally developing CTL populations stimulated with TNP-modified or HSV-infected allo-H-2Kb-bearing cells displayed an allorestricted pattern of recognition. It was further evident that the estimated frequencies of splenic precursors that generated allorestricted CTL clones was two- to threefold higher than the estimated frequencies of precursors that gave rise to the respective alloreactive CTL populations.