Susceptibility to multiple sclerosis is higher in females than males. However, the underlying mechanism behind this gender difference is poorly understood. Because the presence of neuroantigen-primed T cells in the CNS is necessary to initiate the neuroinflammatory cascade of multiple sclerosis, we first investigated how these T cells interacted with astroglia, major resident glial cells of the CNS. Interestingly, we found that myelin basic protein (MBP)-primed T cells from female and castrated male mice, but not from male mice, produced proinflammatory molecules, such as NO, IL-1beta, and IL-6 in astroglia, and these responses were purely via contact between T cells and astroglia. Because T cell:glia contact requires several integrin molecules, we examined the involvement of integrins in this process. Both alpha4 and beta1, subunits of VLA-4 integrin, were found to be necessary for T cell contact-induced generation of proinflammatory molecules in astroglia. Interestingly, the expression of beta1, but not alpha4, was absent in male MBP-primed T cells. In contrast, female and castrated male MBP-primed T cells expressed both alpha4 and beta1. Similarly, we also detected beta1 in spleen of normal young female, but not male, mice. Furthermore, we show that male sex hormones (testosterone and dihydrotestosterone), but not female sex hormones (estrogen and progesterone), were able to suppress the mRNA expression of beta1 in female MBP-primed T cells. These studies suggest that beta1, but not alpha4, integrin of VLA-4 is the sex-specific molecule on T cell surface, and that the presence or absence of beta1 determines gender-specific T cell contact-mediated glial activation.