Thyroid function and prolactin (PRL) responsiveness to thyrotropin-releasing hormone (TRH) were examined in 6 patients with Klinefelter's syndrome prior to and after therapy with testosterone. The thyroid function tests, including serum triiodothyronine (T3), thyroxine (T4), thyroxine binding globulin (TBG), resin T3 uptake (RT3U), radioactive iodine uptake (RAIU), thyrotropin (TSH) stimulation and the TSH response to TRH were normal during both periods of study. Testosterone treatment had no significant effect on any of these parameters with the exception of the RT3U which increased. PRL response to TRH were significantly higher than those observed in normal men (P less than 0.05). Despite the fact that mean plasma PRL responses to TRH were decreased when the patients were restudied during testosterone therapy, they remained greater than those of normal men. Mean serum estradiol concentrations were normal and did not increase significantly during testosterone therapy. These studies suggest that: (1) thyroid function may be normal in patients with Klinefelter's syndrome more often than previously reported, and (2) patients with Klinefelter's syndrome may manifest PRL hyper-responsiveness to TRH that is decreased but not normalized during testosterone therapy. Because estradiol levels failed to increase despite a marked rise in testosterone, further studies are warranted to examine testosterone and estradiol clearance and conversion rates in patients with Klinefelter's syndrome.