Assays were developed to detect and measure autoantibodies (AA) to thyroglobulin (Tg) and to the thyroid hormones, thyroxine (T4) and triiodothyronine (T3). An ELISA to detect AA to Tg was developed, using purified canine Tg as the antigen and goat anti-canine IgG conjugated with alkaline phosphatase as the second antibody. A highly charged agarose electrophoresis assay was used for determination of AA to T4 and T3. Sera from dogs (n = 119) with clinical signs consistent with hypothyroidism were tested for AA to Tg, T4, and T3. Autoantibodies to at least 1 of the 3 thyroid antigens were detected in 58 of the 119 (48.7%) sera tested. Autoantibodies to Tg were detected more frequently in samples with low serum concentrations of thyroid hormones than in samples with normal concentrations. The presence of AA to T4, T3, or both was not significantly associated with low thyroid hormone concentrations, but this lack of association may have been attributable to binding of AA in the measurement of thyroid hormones by radioimmunoassay.