Thyroid hormones exert a permissive influence on the ability of cells to respond to other hormones. In hypothyroidism, stimulation of adenylate cyclase by beta-adrenergic agonists is impaired in rat fat cells, whereas inhibition by adenosine is potentiated. The effects of thyroid status on steady-state levels of the G-protein subunits alpha-Go, alpha-Gi, and beta-G35/36 were investigated using specific antibodies and quantitative immunoblotting of rat fat cell membranes. The amount of alpha-Go (Mr 39,000, alpha-G39) detected in fat cell membranes of euthyroid rats was 44 +/- 5 pmol/mg of membrane protein (n = 5). In the hypothyroid state, the amount of the alpha-subunits of Gi (Mr 41,000, alpha-G41) and Go were found to be markedly increased in comparison to the control. The steady-state level of alpha-G41 and alpha-G39 increased more than 50 and 70%, respectively, in the hypothyroid state. The beta-subunit of G-proteins of rat fat cells appears as a doublet of proteins with Mr = 35,000/36,000 on sodium dodecyl sulfate-polyacrylamide gels. The amount of beta-G35/36 detected in fat cell membranes of euthyroid rats was 0.20 +/- 0.03 nmol/mg of protein (n = 5) and was found to increase by about 60% in the hypothyroid state. Administration of triiodothyronine in vivo (short term hyperthyroidism) resulted in a decrease in the amounts of alpha-G41 and alpha-G39 subunits (25 and 20%, respectively). In contrast to these effects of thyroid hormones on Go and Gi, the steady-state level of beta-adrenergic receptors was not significantly altered by changes in thyroid status. Thus, thyroid status in vivo can modulate the steady-state levels of specific G-proteins.