The present studies have made use of in vitro derived H-2Kb mutants to analyze the fine specificity of alloreactive cytotoxic T lymphocytes (CTL). The variants were derived by negatively selecting mutagenized tumor cells with a monoclonal anti-H-2Kb antibody and positively selecting for residual cells expressing serologically altered H-2Kb molecules. Details of this procedure are described in the companion paper. Selected populations of bulk alloreactive and cloned CTL were examined for recognition of the variants. In contrast to the serologic findings presented in the companion paper, there does not appear to be a correlation between the monoclonal antibody used to select the R8 variant and the CTL specificities recognized. In several instances, CTL clones could discriminate between variants having identical serologic profiles. Therefore, it would appear that the CTL have a large repertoire of allorecognition, even when generated across a mutant anti-Kb combination reflecting only a few amino acid differences. In addition, a diverse set of epitopes can be recognized on the Kb molecule. Finally, in some instances a change in what would appear to be a single amino acid resulted in a profound alteration of CTL recognition even though the Kb mutant molecule expressed limited serologic changes. These results support the idea that small changes in the H-2Kb molecule can have dramatic effects on CTL even though there are relatively little effects on serologic recognition of the target molecule.