The induction of autoimmunity by viruses has been attributed to numerous mechanisms. In mice, coxsackievirus B4 (CB4) induces insulin-dependent diabetes mellitus (IDDM) resembling the final step of disease progression in humans. The immune response following the viral insult clearly precipitates IDDM. However, the molecular pathway between viral infection and the subsequent activation of T cells specific for islet antigen has not been elucidated. These T cells could become activated through exposure to sequestered antigens released by damaged beta cells, or they could have responded to factors secreted by the inflammatory response itself. To distinguish between these possibilities, we treated mice harboring a diabetogenic T cell repertoire with either the islet-damaging agent streptozotocin (STZ) or poly I:C, which nonspecifically activates T cells. Significantly, only treatment of mice with STZ resulted in IDDM and mimicked the effects observed following CB4 infection. Furthermore, antigen-presenting cells from STZ-treated mice were shown to directly activate autoreactive T cells and induce diabetes. Therefore, the primary role of CB4 in the precipitation of IDDM is to damage tissue, causing release and presentation of sequestered islet antigen. These events stimulate autoreactive T cells and thereby initiate disease.