Hyaluronan (HA) is a glycosaminoglycan composed of N-acetylglucosamine and glucuronic acid subunits. Previous studies have suggested that CD44 expressed by T cells bind exogenous HA for their proliferation. However, HA endogenously synthesized by T cells may participate in their autocrine proliferation. In this study, we examined the role of endogenous HA in T cell proliferation using the highly specific HA synthase inhibitor, 4-methylumbelliferone (4-MU). We found that 4-MU inhibited the mitogen-induced synthesis of HA by T cells. Moreover, 4-MU inhibited T cell proliferation in a dose-dependent manner when cells were cultured with different stimuli, including Con A, PMA/ionomycin, and allogeneic spleen cells. Furthermore, 4-MU inhibited mitogen-stimulated IL-2 secretion, suggesting that HA may play a role in the production of this cytokine. Addition of IL-2 to T cells treated with 4-MU and Con A reversed the block in cell proliferation, showing that impaired IL-2 production is a likely mechanism for the inhibited division of T cells. Surprisingly, an anti-CD44 Ab antagonistic for HA binding did not reduce IL-2 secretion or T cell proliferation. Importantly, 4-MU did not alter the surface expression of CD44 or the ability of CD44 to bind to HA. Thus, HA-mediated IL-2 production and T cell proliferation are CD44 independent. Our results strongly suggest that HA synthesized by T cells themselves is critical for their IL-2-mediated proliferation and have revealed a previously unrecognized role for endogenous HA in T cell biology.