Exposure of female Holtzman rats to constant light (24 hr/day) immediately after birth significantly increased 9,10-dimethyl-1,2-benzanthracene-induced mammary cancer. Such “functionally pinealectomized” animals also revealed significant increase in the circulating level of prolactin and exaggerated development and proliferative activity of mammary epithelium, as measured by quantitation of terminal end buds and alveolar buds from the whole mounts and by DNA synthesis, respectively. Administration of melatonin (500 µg/day/rat i.p. given from 52 to 145 days of age) completely abolished the effect of functional pinealectomy by sharply reducing 9,10-dimethyl-1,2-benzanthracene-induced cancer incidence from 95% to 25% during the post-9,10-dimethyl-1,2-benzanthracene observation period which lasted up to 180 days. On the other hand, administration of melatonin to surgically pinealectomized animals exposed to constant light reversed the effect only partially by reducing the cancer incidence from 83% to 53%. Further, melatonin treatment in intact and surgically pinealectomized animals exposed to a short photoperiod revealed qualitatively similar differences in suppression of the cancer incidence. From these results, it is concluded that, to have an impressive antitumor effect, presence of the pineal gland is essential, and the probable site of melatonin action appears to be at both the pineal gland and the hypothalamus.