Bacille Calmette-Guérin (BCG) immunity can be studied as one experimental model for mycobacterial protective immunity. We have used flow cytometry to investigate human T cell subsets induced by BCG vaccination. PBMC harvested from BCG-vaccinated individuals and controls were stimulated with mycobacterial Ags, and the T cell subsets present after 7 days of in vitro expansion were characterized. The most dramatic expansions induced by mycobacterial Ags were detected in gamma delta T cells. The gamma delta T cell expansions measured after in vitro stimulation with mycobacterial Ags were significantly greater in BCG responders compared with nonsensitized controls, indicating that BCG vaccination induced gamma delta T cell activation associated with enhanced secondary responses. The majority of gamma delta T cells induced by BCG vaccination were gamma 9+ delta 2+ T cells reactive with isoprenyl pyrophosphates. Coculture with CD4+ T cells induced optimal gamma delta T cell expansion, although IL-2 alone could provide this helper function in the absence of CD4+ T cells. Gamma delta T cells were found to provide helper functions for mycobacterial specific CD4+ and CD8+ T cells as well, demonstrating reciprocal stimulatory interactions between gamma delta T cells and other T cell subsets. Finally, prominent mycobacterial specific gamma delta T cell expansions were detected in a subset of unvaccinated controls with evidence for prior sensitization to mycobacterial lysates (elevated mycobacterial specific lymphoproliferative responses). These latter findings are consistent with the hypothesis that exposure to atypical mycobacteria or related environmental Ags may induce gamma delta T cells cross-reactive with Ags present in the Mycobacterium tuberculosis complex. Our results suggest that gamma delta T cells may be capable of developing a memory immune-like phenotype, and therefore might be important targets for new vaccines.