The importance of Ag-specific gamma delta T lymphocytes in human immune responses to pathogenic organisms is unknown. In the present study the expression of gamma delta TCR on T lymphocytes from patients with cutaneous, mucosal, or visceral leishmaniasis was examined. All of these patient groups had elevated levels of gamma delta T cells in peripheral blood. Patients' gamma delta T cells included CD8+ as well as null cells. The percentage of T cells expressing gamma delta TCR was increased significantly by stimulation in vitro with certain parasite Ag. T-cell lines generated by stimulation with promastigote lysates of Leishmania amazonensis or L. braziliensis typically contained 25 to 60% gamma delta T cells. In contrast, two immunodominant surface Ag of L. amazonensis, gp63 and gp42, did not expand gamma delta T cells from infected patients, although both Ag elicited strong alpha beta T-cell responses. gamma delta T cells isolated from a Leishmania-specific T-cell line responded to stimulation with promastigote lysate. Of particular interest, gamma delta T cells from PBMC of a patient with mucosal leishmaniasis responded to stimulation with a recombinant 70 kDa heat shock protein of L. chagasi. This study demonstrated that several clinical forms of leishmaniasis induced elevated numbers of gamma delta T cells that responded specifically to Leishmania Ag in vitro. Therefore, this component of the T-cell response to Leishmania may impact the outcome of clinical disease.