The OX-40 receptor, a member of the nerve growth factor/tumor necrosis factor receptor gene family, is expressed preferentially on autoreactive CD4+ T cells isolated from the site of inflammation in rats with clinical signs of experimental autoimmune encephalomyelitis (EAE). To examine whether the OX-40 receptor has biologic relevance to T cell function, we evaluated the ability of a rat OX-40 receptor-specific antibody to co-stimulate a myelin basic protein (MBP)-reactive CD4+ T cell line. The anti-OX-40 antibody provided a potent co-stimulatory signal to CD4+ T cells when added in conjunction with a submitogenic dose of anti-CD3, but the anti-OX-40 antibody alone did not produce a mitogenic response. The magnitude and dose-response of anti-OX-40 co-stimulation was virtually identical to the signal delivered to T cells when cultured with anti-CD28 in conjunction with anti-CD3. MBP-specific T cells stimulated with both anti-CD3 and anti-OX-40 antibodies expressed increased mRNA and protein for IL-2 when compared to anti-CD3 alone. MBP-specific T cells stimulated with both anti-CD3 and anti-OX-40 antibodies were also able to induce EAE when transferred into naive Lewis rats. In contrast, cells stimulated with anti-CD3 alone were not encephalitogenic. These data suggest that the function of the OX-40 receptor on activated T cells is to provide an alternative pathway for T cell co-stimulation that may be similar in potency to the CD28-mediated signal.