The 64,000-M(r) (64K) islet autoantigen, which is considered to be a target protein of beta cell destruction in insulin-dependent diabetes mellitus (IDDM), has recently been identified as the enzyme glutamic acid decarboxylase (GAD). We reported a two- to three-fold increased expression of the antigen in islets of diabetes-susceptible mice following infection with a diabetogenic strain of Coxsackievirus B4 (CB4) at 72-h postinfection (p.i.), a time point of active virus replication in the islets. Most of the infected animals subsequently developed 64K autoantibodies and hyperglycemia. Since the infection increases 64K expression, we have analysed immunoreactive GAD expression with a panel of peptide antisera and two widely-used polyclonal antisera against GAD, and measured GAD activity in the brain, pancreas and islets of these mice. Two isoforms, GAD65 and GAD67, are detected in these tissues from non-infected mice. Both GADs are also present in the infected mice brain at 72 h p.i.; however, their islets contain about three-fold more GAD65, and essentially no detectable GAD67. GAD activity is significantly higher in the brain compared with whole pancreas or islets, and islet GAD activity is higher than pancreas GAD activity. The infection significantly reduces islet GAD activity, but not brain GAD activity. CB4-induced abnormalities in islet GAD expression may play a role in virus-induced diabetes.