Cats are the only nonhuman mammalian species with a high incidence of hyperthyroidism, and a better understanding of the pathogenesis of feline hyperthyroidism is of clinical relevance for veterinary medicine. The etiology of this disease in cats remains controversial. Both an intrinsic autonomy of growth and function of follicular cells as well as an autoimmune-related mechanism have been proposed. To explore the role of the autologous TSH receptor (TSHR) in the pathogenesis of hyperthyroidism in cats, we cloned the coding sequence of the feline TSHR by RT-PCR. The open reading frame consists of 2292 nucleotides and encodes a 763-amino acid protein, one amino acid less than the human TSHR. Species comparison reveals that the cat TSHR is most closely related to the canine TSHR, with 96% identity and 97% similarity in amino acid sequence. cAMP accumulation, inositol phosphate production, and TSH binding were similar in the feline TSHR, compared with the human receptor. Analogous to the human TSHR, the cat TSHR also displays basal constitutive activity. To test the possibility that hyperthyroid cats develop antibodies that stimulate the autologous receptor, transfected cells expressing the feline TSHR were treated with sera or purified IgG obtained from 16 hyperthyroid cats. There was no increase in cAMP-dependent luciferase activity in the hyperthyroid cats, suggesting the absence of stimulatory autoantibodies. These sera were also negative for TSH-binding inhibitory Igs in an RRA. At least in the animals included in this study, there is no evidence for the presence of circulating thyroid stimulating factors as a mechanism underlying the pathogenesis of feline hyperthyroidism, and the findings support a model involving intrinsic autonomy of thyroid follicular cell growth and function.