Recent results suggested that a large subset of heat shock protein HSP-60 reactive peripheral lymphoid gamma delta T cells preexists in normal adult mice, all members of which respond to a single segment of this common HSP. However, the experimental evidence supporting this idea involved in vitro peptide responses of gamma delta T-cell hybridomas generated from unprimed spleen cells. Here, we report an attempt to elicit a gamma delta T-cell response in vivo by stimulation of adult C57BL/10 mice with HSP-60 or an HSP-60-derived peptide fragment comprising amino acids 180-196 of mycobacterial HSP-60. Whereas no gamma delta T-cell response was detectable in mice injected with the intact protein, stimulation with the peptide altered the reactive gamma delta T-cell population in vivo. These changes were detected among hybridomas generated with cells restimulated in vitro and included a large increase in hybridizable gamma delta T cells, a nearly maximal increase in the relative frequency of HSP-60-reactive cells, and structural changes in expressed T-cell receptors of HSP-60-reactive cells. Interestingly, we failed to elicit a detectable alpha beta T-cell response to the particular peptide stimulatory for gamma delta T cells, although at least three other HSP-60 epitopes were recognized. Our data show that normal gamma delta T cells can respond in vivo to small peptide antigens. The gamma delta T-cell response to the HSP-60-derived peptide studied here is apparently independent of antigen-specific alpha beta T-cell reactivity.