AbstractAs a part of our continuing effort to understand the nature of the inhibition of thyroxine‐induced metamorphosis by prolactin in the tiger salamander, we examined the effects of delaying the initiation of prolactin treatment for progressively longer time intervals following the injection of a metamorphosing dose of thyroxine. Prolactin continued to inhibit gill resorption when given either 2 or 4 days following thyroxine, but the magnitude of the inhibition appeared to diminish. Delay of prolactin treatment until day 6 abolished the inhibition. In contrast, tail fin regression appeared to be inhibited in an undiminished fashion by prolactin given 2, 4, or 6 days following thyroxine. Body weight loss was inhibited in a similar manner. In a second experiment, actinomycin D, a transcription inhibitor, was observed to inhibit metamorphic changes. A delay of 5 days in the administration of this drug relative to thyroxine treatment produced a significant reduction in its inhibition of gill resorption, tail fin regression, and body weight loss. Surprisingly, late (day 5 following thyroxine) treatments with prolactin still produced a significant inhibition of fin regression and weight loss even when the inhibitory dose of actinomycin D was given on day 5 along with the prolactin. These results are discussed with respect to their implications concerning our understanding of the mechanism of prolactin action during amphibian metamorphosi