T cell mediated responses play a role in xenograft as well as allograft rejection. In vitro T cell responses to xenogeneic antigens are characterized by T cell recognition of polymorphic determinants of gene products encoded by the major histocompatibility complex of the stimulating cells, but little is known of the fine specificity of this recognition of xenogeneic antigens and its comparability to allogeneic antigen recognition. In order to study the fine specificity of human CTL in the recognition of xenogeneic antigens, long-term lines or clones were established from a secondary mixed lymphocyte response in which the stimulator cells were H-2b murine splenocytes. By comparing the ability of a series of target cells derived from congenic recombinant mouse strains to be lysed by these CTL, it was demonstrated that all isolated lines specifically lysed target cells expressing H-2b or T1a/Qa-1b products. Of the effector populations specific for H-2b cell surface molecules, all recognized class I products with Kb specificity predominating when evaluated for their ability to lyse in vivo-derived class I mutants or cells transfected with class I genes. These human xenoreactive CTL were able to distinguish wild type Kb molecules from those altered by amino acid changes confined to a single molecular domain of Kb. These findings demonstrate that xenogeneic antigen recognition by human T cells is characterized by a fine specificity of antigen recognition comparable to the specificity of recognition of major histocompatibility complex-encoded molecules by murine CTL across allogeneic differences.