The effects of a thyroidectomy and thyroxine (T 4 ) replacement on the spontaneous and human chorionic gonadotropin (hCG)-stimulated secretion of testosterone and the production of adenosine 3Π,5Π-cyclic monophosphate (cAMP) in rat testes were studied. Thyroidectomy decreased the basal levels of plasma luteinizing hormone (LH) and testosterone, which delayed the maximal response of testosterone to gonadotropin-releasing hormone (GnRH) and hCG in male rats. T 4 replacement in thyroparathyroidectomized (Tx) rats restored the concentrations of plasma LH and testosterone to euthyroid levels. Thyroidectomy decreased the basal release of hypothalamic GnRH, pituitary LH, and testicular testosterone as well as the LH response to GnRH and testosterone response to hCG in vitro. T 4 replacement in Tx rats restored the in vitro release of GnRH, GnRH-stimulated LH release as well as hCG-stimulated testosterone release. Administration of T 4 in vitro restored the release of testosterone by rat testicular interstitial cells (TICs). The increase of testosterone release in response to forskolin and androstenedione was less in TICs from Tx rats than in that from sham Tx rats. Administration of nifedipine in vitro resulted in a decrease of testosterone release by TICs from sham Tx but not from Tx rats. The basal level of cAMP in TICs was decreased by thyroidectomy. The increased accumulation of cAMP in TICs following administration of forskolin was eliminated in Tx rats. T 4 replacement in Tx restored the testosterone response to forskolin. But the testosterone response to androstenedione and the cAMP response to forskolin in TICs was not restored by T 4 in Tx rats. These results suggest that the inhibitory effect of a thyroidectomy on the production of testosterone in rat TICs is in part due to: 1) the decreased basal secretion of pituitary LH and its response to GnRH; 2) the decreased response of TICs to gonadotropin; and 3) the diminished production of cAMP, influx of calcium, and activity of 17β€-HSD. T 4 may enhance testosterone production by acting directly at the testicular interstitial cells of Tx rats.