Breeding in the three-spined stickleback is stimulated by long but not by short photoperiods in many seasons. The aim of the present study was to test the hypothesis that melatonin plays a role in the inhibitory effect of short photoperiod in this species. Adult nonbreeding males were kept either under constant light (Experiment 1) or under a stimulatory long photoperiod (16L 8D, Experiment 2), in water containing 0, 20, or 80 microg/liter melatonin for 16 hr/day for 28 days during the spring. These melatonin treatments were intended to simulate the daily melatonin pattern of a nonstimulatory short photoperiod. In the second experiment, fish were also kept under a nonstimulatory short photoperiod (8L 16D). In the natural breeding season the only germ cells found in the stickleback testes are spermatozoa and spermatogonia, a condition found in many fish under all treatments. In the first experiment, spermatogenesis was not influenced by melatonin. However, testes also containing spermatocytes and spermatids were more common in fish kept under 8L 16D and fish treated with 80 microg/liter melatonin than in 16L 8D controls in Experiment 2. Kidney hypertrophy, an androgen-dependent male secondary sexual characteristic in the stickleback, appeared in most males kept under constant light or 16L 8D and was not influenced by melatonin treatment. In contrast, control males kept under 8L 16D in Experiment 2 did not display kidney hypertrophy. Therefore, the presence of an extended period of elevated melatonin did not prevent the stimulatory effects of long photoperiod on development of this secondary sexual characteristic in the stickleback.