The AKR.H-2bSL1 tumor cell line is susceptible to H-2Kb-restricted cytotoxic T lymphocytes (CTL) directed against the subclass of AKR endogenous leukemia virus-induced tumors that express the Gross cell surface antigen (anti-AKR/Gross virus CTL). A variant subclone (cl.18-5) of AKR.H-2bSL1 was isolated, whose susceptibility to lysis by conventional or cloned lines of anti-AKR/Gross virus CTL was approximately 5% or less than that of the parental tumor. The cl.18-5 variant was also ineffective when used as an in vivo priming cell or an in vitro stimulator cell in the generation of anti-AKR/Gross virus CTL or as an unlabeled target cell in competitive inhibition assays. These results implied that the failure of cl.18-5 to be lysed was due to a lack of recognition by the CTL. In contrast, cl.18-5 was able to be lysed by and stimulate the generation of predominantly H-2Db-restricted CTL with apparent specificity for AKR minor histocompatibility antigens. The variant line was also about as susceptible as the parental AKR.H-2bSL1 line to both allogeneic CTL and to H-2Kb-restricted, TNP-specific CTL. Thus, the lack of recognition of cl.18-5 by anti-AKR/Gross virus CTL did not appear to be due to a failure to express functional H-2 products or to a generalized insusceptibility to H-2-restricted CTL. Rather, cl.18-5 appeared to be a selective variant and a useful probe for studying the specificity of anti-AKR/Gross virus CTL.