Diabetogenic Coxsackievirus B4 infection produces a diabetes syndrome in susceptible mice resembling insulin-dependent diabetes mellitus. We reported a two- to threefold increased expression of a 64,000-Mr (64 K) islet autoantigen in the infected mice preceding the development of hyperglycemia, suggesting a possible role of the virus in autoimmunity. To assess if the virus could be a trigger of autoimmunity, 64 K autoantibody development was correlated with hyperglycemia and virus replication in islets during early and late infection. Additionally, the effects of blood removal from these mice on the incidence of hyperglycemia and antibody production were evaluated. Noninfected control mice were essentially 64 K antibody negative, the infected consistently positive. Approximately 30% of the animals developed antibodies by 72 h postinfection (p.i.) and 90% by 4-6 wk p.i. Virus-induced hyperglycemia appeared bimodal: hyperglycemia in 50% of the mice by 1 wk p.i., which decreased to 30% by 3 wk and then increased to 80-100% by 6 wk p.i. Infectious virus was abundant in the islets at 72 h but undetectable later. Hyperglycemia seen at 6 wk decreased dramatically (67-73%) if the mice were bled once between 72 h and 2 wk p.i. Only 50-60% of the mice bled once were 64 K positive compared to 90% positive nonbled mice. Coxsackievirus may initiate or enhance the autoimmune response.