The relationship of serologically defined determinants to determinants recognized by cytotoxic T cells on molecules encoded by the Kk gene of the murine major histocompatibility complex (H-2) has been analyzed. For this purpose we used three somatic variants of a Kk-expressing lymphoma line lacking individual determinants of the Kk molecule, as defined by monoclonal antibodies (mAb), as target cells for Kk-specific alloreactive and Kk-restricted cytotoxic T lymphocytes (CTL) cloned by limiting dilution. Neither alloreactive nor fluorescein isothiocyanate, influenza- or Newcastle disease virus-specific Kk-restricted CTL clones were found to distinguish between variants and wild type cells, indicating that the serologically defined determinants lost by the variants were not essential for antigen recognition of CTL with these specificities. On the other hand, two of the variants lacking either one of a pair of serological determinants were discriminated from Kk wild type cells by about 40% of Kk-restricted, trinitrophenol (TNP)-specific CTL clones. The third variant, lacking both of the determinants, however, was lysed by all CTL clones to the same extent as wild type cells. From these results we conclude that the determinants restricting the TNP-specific CTL were also not identical with those defined by mAb. In experiments performed to optimize the conditions for the limiting dilution analysis we found that the specificity of the CTL stimulation was strongly dependent on the concentration of T cell growth factor (interleukin 2) in the cultures during CTL stimulation. High concentrations of IL2 resulted in a drastic increase in the frequency of CTL clones. Part of these clones, however, were found not to be specific for antigens present on the stimulator cells.