Oral administration of Ag, over a period of several days, induces a state of tolerance that is associated with activation of CD8+ T cells that can transfer unresponsiveness to naive syngeneic hosts. We previously demonstrated that these T cells are not CTL precursors and that they inhibit responses by CD8+ CTL, as well as Ab and CD4+ T cell responses. Activation of noncytolytic, CD8+ suppressor T cells by oral Ag is a process that is not understood. In these studies, we asked whether depletion of the gamma delta T cells altered induction of oral tolerance. Injection of the anti-delta-chain Ab (GL3) down-modulated the expression of gamma delta TCR and inhibited the induction of oral tolerance to OVA, as measured by Ab, CD4+, and CD8+ T cell responses. GL3 did not activate IL-2 secretion that could be detected in the serum, nor did it induce IL-2R expression by intraepithelial lymphocytes, suggesting that GL3 inhibited the function of gamma delta T cells rather than activating them. This interpretation is supported by our observation that oral administration of Ag did not induce tolerance in TCR-delta knockout mice. These data suggest that gamma delta T cells play a critical, active role in tolerance induced by orally administered Ag.