We reported that antigenic preparations from Yersinia enterocolitica stimulate murine T cells in a manner consistent with that of superantigens. As a consequence we examined whether Y. enterocolitica antigenic preparations stimulate human T-cell cultures. Human T cells, enriched from peripheral blood lymphocytes, were stimulated to proliferate in the presence of Y. enterocolitica cytoplasmic and membrane preparations. This activity has also been shown to be sensitive to protease treatment, indicating the presence of a protein, and when separated by ion-exchange chromatography a single peak of activity is resolved. Furthermore, this proliferation was inhibited, in a dose-dependent manner, by the presence of antibodies directed against MHC class II antigens, indicating a requirement for these molecules. When these cells were stained with a panel of V beta-specific antibodies to determine if there was an enrichment of a particular V beta-bearing T-cell subset after stimulation, results indicate a significant enrichment of T cells bearing V beta 3, V beta 12, V beta 14, and V beta 17 over controls. Taken together, these data are consistent with a Y. enterocolitica product acting as a superantigen for human T cells.