Melatonin administered in vivo had previously been shown to inhibit thyroid cell proliferation and subsequent in vitro thyroxine (T4) secretion in anuran tadpoles. Melatonin in vitro also directly reduced the sensitivity of the thyroid to thyrotropin (TSH). The present work sought to determine whether melatonin directly affected baseline, unstimulated T4 secretion, and to compare its effect with that of prolactin (PRL). Thyroids from larval Rana catesbeiana or adult Rana pipiens were incubated in control or melatonin (0.01 to 100 μg/ml) media. Melatonin directly inhibited T4 secretion by thyroids from both tadpoles and frogs at all concentrations of melatonin used and at both prometamorphic and climax tadpole stages. PRL, used in vitro at 10 μg/ml, did not influence the response of the thyroid to TSH (0.2 μg/ml) in young tadpoles, or the baseline secretion of T4 by thyroids at any stage of larval life except climax, when T4 secretion was significantly decreased by the third day of culture. Thus although both melatonin and PRL have been shown to antagonize the action of T4 in vitro, and to decrease metamorphic rate, melatonin is a much more effective thyroid gland inhibitor than PRL. J. Exp. Zool. 286:625–631, 2000. © 2000 Wiley‐Liss, Inc.