After infection with Coxsackievirus B3 (CB3), H-2 congenic strains of mice on an A-background develop an immunologically mediated myocarditis associated with autoantibodies directed mainly against cardiac myosin. We tested these autoantibodies for cross-reactivity with the virus. Using immunoblotting and virus neutralizing assays with affinity purified antibodies and absorbed sera, we found that the autoantibodies to heart myosin did not cross react with CB3. In addition, myosin autoantibodies induced by immunization with cardiac myosin did not react with the virus. Western immunoblotting revealed that the anti-CB3 antibodies in the infected mice are directed against the capsid protein VP 2. We conclude that CB3 antigens do not stimulate an immune response that cross-reacts with cardiac myosin.