Synthesis and release of melatonin were studied in pineal explants from 14- (young) and 60-day-old ('maturing') male Long-Evans rats with or without added thyroid hormone, triiodothyronine (T3), at or near physiological levels and under light and dark conditions. Incubation for 6 hr (1200-1800) was in a synthetic medium; melatonin was measured by radioimmunoassay (RIA). In light, T3 increased melatonin levels in pineal and medium of cultures from either young or maturing animals. In dark, T3 decreased melatonin levels in the pineals of either age, but was without significant effect on levels in the medium. Since it is known from other work that 14-day-old rat pineal glands do not yet have a complete sympathetic innervation system, it is here doubly evident that T3 can modulate directly pineal synthesis and release of melatonin, and may not depend upon a mature sympathetic innervation. Light in the studied conditions was permissive from the stimulatory action of T3 on pineal synthesis and release of melatonin in vitro.