Although low doses of tumor-derived stress protein gp96 elicit protective immunity to the tumor from which it is isolated, protection is lost at high doses because of the induction of immunoregulatory CD4 T cells. This study evaluated the influence of gp96 on resting rat bone marrow–derived dendritic cells (BMDCs) and purified CD3 T cells. In contrast to previous reports, gp96 had no effect on adhesion and costimulatory molecule expression by BMDCs, nor did it influence interleukin (IL)-10 and IL-12 secretion or their allostimulatory capacity. Gp96 did not bind to BMDCs but dose-dependently bound to CD4 and CD8 T cells. At low concentrations (1 and 25 μg/mL), gp96 acted as a costimulator of CD3 T cells, inducing proliferation and the secretion of interferon (IFN)-γ and IL-10. Gp96 also increased the proliferation of CD28-costimulated CD3 T cells and their secretion of IFN-γ, IL-4, and IL-10. Gp96 had no effect at higher concentrations (50 and 100 μg/mL), despite the occurrence of cell surface binding at these concentrations. These findings indicate that gp96 can act as a costimulatory molecule for CD3 T cells, and an observed increase in the IL-10:IFN-γ secretion ratio induced by gp96 suggests that it might, at appropriate concentrations, promote a regulatory T-helper 2 (Th2)–like phenotype.