The zoological specificity of human thyroid-stimulating antibody (TSAb) that occurs in the blood in Graves' disease was examined by assessing its effect on the thyroid of the dog, guinea pig, calf, and mouse as well as that of man. With all but murine glands, thyroid stimulation was assayed by measuring the increase in the concentration of cAMP in the thyroid slices or fragments after 2 h of incubation in buffer containing TSAb. Effects on the thyroid of the mouse were monitored by the in vivo bioassay for LATS. Sera from 33 patients with Graves' disease were obtained and concentrates of TSAb were prepared by precipitation of IgG with 1.64 M (NH4)2SO4. These all stimulated the human thyroid, 13 were LATS-positive, and they variably affected the tissues of other species; of 27 tested, 14 stimulated the thyroid of the dog, 8 out of 23 stimulated the thyroid of the guinea pig, and 12 out of 16 stimulated the gland of the calf. The more potent the TSAb as assayed with human tissue, the more likely was it to stimulate other species of thyroid; however, frequent exceptions occurred. In a separate analysis of 35 LATS-positive preparations of TSAb, correlation between the responses in the LATS and human thyroid slice assays was statistically significant (P less than 0.001). The data are compatible with the view that stimulation by TSAb of nonhuman thyroids, including the murine as in the LATS bioassay, reflects cross-reaction of this immunoglobulin with an antigen that has sufficient similarity to the human molecule to be recognized by the human antibody.