Tissue distribution of 125I-thyroxine (T4) and 3H-melatonin and the effect of each hormone on the tissue content of the other were studied because previous work indicated that melatonin antagonized metamorphosis through peripheral, as well as thyroidal effects. Late pre- to prometamorphic Rana catesbeiana tadpoles on an 18 light:6 dark cycle were used for injection of hormones in vivo or to supply tissues for in vitro hormone administration. Labeled melatonin uptake was highest in intestine, ventral skin and pituitary; lowest in thyroid and brain and intermediate in hindlimb, tail and gills. The tissue content of labeled T4 was distributed in nearly the same way, except that the thyroid level was relatively higher, and pituitary lower, than that of labeled melatonin. The pineal, studied only in the tracer T4 experiments, had the highest content of labeled T4 of all tissues. Simultaneous injection of either 0.007 or 0.2 microgram T4 increased 3H-melatonin uptake into peripheral tissues that undergo major metamorphic changes but not into neural or endocrine organs. In contrast, 0.033, 3.75 or 15 micrograms melatonin had no significant influence on the content of 125I-T4 in any tissue studied in vivo. Results of in vitro labeling of selected tissues were generally in agreement with the in vivo work except that the 125I-T4 content of intestinal segments from late prometamorphic larvae was lower in melatonin-treated than in control groups. The results suggest that peripheral tissues are a major site for T4-melatonin interactions and that T4 may modulate its own action through influencing melatonin levels in target tissues and perhaps in the thyroid. Because melatonin had no effect on tissue T4 content in young tadpoles, retardation of metamorphic events by melatonin does not seem to involve modulation of T4 availability to the tissues.