BALB/c mice that are infected with reovirus Type 1 develop thyroiditis. Viral antigens were seen in the cytoplasm of epithelial cells but not in the surrounding colloidal space of the thyroid. Examination of sera from the infected mice revealed autoantibodies that, by immunofluorescence, reacted with second antigens in the colloid (ground-glass staining pattern) and thyroglobulin (puffy staining pattern). An enzyme-linked immunosorbent assay designed to identify the reactive antigens showed the autoantibodies to direct against thyroglobulin. Synthetic serum thymic factor (FTS) suppressed autoantibody production to the thyroid after reovirus Type 1 infection. Reovirus Type 3, in contrast to reovirus Type 1, did not induce autoantibodies to react against thyroglobulin. By the use of recombinants between reovirus Type 1 and Type 3, the segment of the reovirus genome responsible for the induction of autoantibodies to thyroglobulin was identified. Virus containing the S1 genome segment from reovirus Type 1, which codes the sigma 1 polypeptide (i.e. haemagglutinin), infected epithelial cells in the thyroid and induced autoantibodies against the thyroglobulin. However, virus containing the S1 gene segment from reovirus Type 3 failed to infect cells in the thyroid and did not induce autoantibodies against thyroglobulin. In this study, reovirus Type 1 induces thyroiditis and autoimmunity, and the S1 gene segment is required for the induction of autoantibodies against thyroglobulin.