Since it is known that opiates inhibit TSH [thyrotropin] secretion, the effect of Leu-enkephalin on TSH secretion was studied. When 250 .mu.g Leu-enkephalin was injected i.p. into male rats, the mean serum TSH level at 30 min. was 770 ng/ml as opposed to 1134 ng/ml in the saline control group (P < 0.02). To elucidate the mechanism of the TSH-inhibiting activity of Leu-enkephalin a series of in vitro experiments were undertaken with rat hemipituitaries [hp]. This system was 1st validated by demonstrating TSH-releasing activity of TRH [thyroliberin] at various times. The effect of Leu-enkephalin on TRH mediated TSH release was then determined. A suppressive effect was demonstrated (11.7 .+-. 0.9 .mu.g TSH/hp per 3 h in the TRH alone group vs 9.9 .+-. 0.8 .mu.g TSH/hp per 3 h in the TRH plus Leu-enkephalin group, (P < 0.05). Naloxone failed to overcome the TSH-inhibiting activity of Leu-enkephalin (14.1 .+-. 1.2 .mu.g TSH/hp per 3 h for TRH alone vs 9.0 .+-. 0.8 .mu.g/hp per 3 h in the TRH plus Leu-enkephalin plus naloxone group, P < 0.005). Leu-enkephalin suppresses basal TSH in vitro and blocks the TSH response to TRH by a direct pituitary action. The possible physiological significance of these findings is discussed.