Twelve heifers were exposed to 21 degrees ambient temperature for 10 days, and then subjected to 4.5, 21, or 32 degrees for 9 days in controlled environmental chambers. Serum prolactin (PRL) decreased linearly (P less than 0.01; 0.6 ng/ml/degrees) as the temperature was reduced during the first day from 21 to 4.5 degrees; serum PRL increased linearly (P less than 0.05; 1.17 ng/ml/degrees) as the temperature was increased from 21 to 32 degrees. Between Days 2 and 9 serum PRL averaged 2.6, 13.0, and 27.7 ng/ml (P less than 0.05) at 4.5, 21, and 32 degrees, respectively. Injection of thyrotropin-releasing hormone (TRH) caused serum PRL to increase within 5 min from 20.4 to 109.8 ng/ml at 32 degrees, at 21 degrees serum PRL increased from 15.7 to 62.8 ng/ml, whereas at 4.5 degrees serum PRL did not respond to TRH. Serum growth hormone (GH) averaged 4.0, 6.3, and 9.4 ng/ml at 4.5, 21, and 32 degrees, respectively, but these means were not different (P greater than 0.10). TRH released GH at all temperatures tested, but the quantity released was unaffected by ambient temperature. Relative humidities of 50 and 90% did not significantly alter (P greater than 0.05) serum PRL or GH. We conclude that ambient temperature affects basal and TRH-stimulated concentrations of serum PRL but not GH in heifers.