Of more than 6,000 canine serum samples submitted to the Endocrine Diagnostic Laboratory of the School of Veterinary Medicine, Auburn University, Ala, for thyroid evaluation in 1983, 18 contained an abnormal triiodothyronine (T3)-binding factor (T3BF). These samples were easily distinguished from non-T3BF containing samples because the factor interfered with the radioimmunoassay for total T3 resulting in a profound increase in apparent values for T3 concentration. Most of the T3BF-containing samples did not have unusual thyroxine binding or inappropriate thyroxine concentrations. Triiodothyronine binding in samples containing T3BF was inhibited by addition of exogenous T3 and was, with one exception, not affected by the addition of 8-anilino-1-naphthalene sulfonic acid (ANS). Apparent affinity constants determined by Scatchard analysis were considerably greater than those of circulating T3-binding proteins found in human beings, but were similar to values obtained for human T3 autoantibodies. After the addition and incubation of [125I] T3 with T3BF-containing samples, a large fraction of the radioactivity bound by T3BF could be precipitated by the addition of goat antibody to dog immunoglobulin G. Additional observations that the binding was insensitive to inhibition by ANS, was resistant to heat inactivation, and had a high apparent affinity constant for T3 were consistent with the idea that the factor was an autoantibody to T3.