Experimental autoimmune encephalomyelitis (EAE) is a widely used animal model of multiple sclerosis (MS), a human autoimmune disease. To explore how EAE and ultimately MS is induced, autoantigen-specific T cells were established, were labeled with fluorescent protein by retroviral gene transfer, and were tracked in vivo after adoptive transfer. Intravital imaging with two-photon microscopy was used to record the entire entry process of autoreactive T cells into the CNS: a small number of T cells first appear in the CNS leptomeninges before onset of EAE, and crawl on the intraluminal surface of blood vessels, which is integrin α4 and αL dependent. After extravasation, the T cells continue into the perivascular space, meeting local antigen-presenting cells (APCs), which present endogenous antigens. This interaction activates the T cells and guides them to penetrate the CNS parenchyma. As the local APCs in the CNS are not saturated with endogenous antigens, exogenous antigens stimulate the autoreactive T cells more strongly and, as a result, exacerbate the clinical outcome. Currently, we are attempting to visualize T-cell activation in vivo in both rat T-cell-mediated EAE and mouse spontaneous EAE models.