An enzyme-linked immunosorbent assay was established to detect autoantibodies in canine patients with hypothyroidism. Thyroglobulin, purified from thyroid glands of euthanized, healthy dogs, was used as antigen. Utilizing this test system, sera of 39 patients with hypothyroidism were screened for the presence of thyroglobulin specific autoantibodies. Positive titers were found in 38% of these dogs, whereas 18 out of 72 patients (25%) with other internal diseases showed a positive reaction. In addition 21 healthy dogs were tested for the presence of anti-thyroglobulin autoantibodies. Three dogs were found to be positive in the ELISA. Development of autoantibody titers was followed up in four cases over a period of one year. A steady decline of autoantibody titers was observed in three patients with hypothyroidism, whereas titers of one healthy dog remained high over this period.