The purpose of this work was twofold: 1) to learn whether rats transgenic for HLA-B27 and the human beta 2-microglobulin gene HB2M can mount B27-restricted cytolytic T lymphocyte (CTL) responses to the male H-Y antigen, and 2) to learn whether such CTLs would recognize both rat and mouse H-Y in the context of HLA-B27. Female rats of the B27/HB2M transgenic line 21-4L were primed in vivo with cells from males of the same line. CTL effectors were generated from lymph node cells of these females following culture with irradiated antigen-presenting cells from either male 21-4L rats or male mice of the B27/HB2M transgenic 56-3 line. The CTLs showed male-specific, B27-specific lysis of both rat and mouse targets. Lysis of B27 targets was inhibitable by monoclonal antibodies specific for B27 or rat CD8. Specific lysis of male B27 rat and mouse targets was inhibitable equally by either rat or mouse male B27 cold targets, but not significantly by female or nontransgenic cold targets. The B27-restricted CTLs neither recognized nor were inhibited by B27+ or B27- male or female human targets. These results demonstrate that CD8+, B27-restricted, anti-H-Y CTLs recognize an evolutionarily conserved H-Y peptide antigen in both rats and mice. In addition, they establish the transgenic rat as a model system for examining the T-cell response to antigen presented by class I HLA molecules.