Immature male Wistar rats with a 12:12 h light-dark cycle received daily s.c. melatonin injections of 100 micrograms from 20 to 40 days of age. Melatonin administration during the late photophase (9 h after the onset of light) or during the late scotophase (9 h after the onset of darkness) caused reduced weights of testes and seminal vesicles, lowered plasma levels of testosterone, LH and FSH, and decreased number of pituitary GnRH receptors, a series of observations which reflects delayed sexual maturation at 40 days. In contrast, no effect was observed when melatonin was injected during the early photophase of scotophase (5 h after the onset of light or darkness, respectively). In order to better investigate the night hours, melatonin action was studied in rats that were born and raised with a shifted 12:12 h light-dark cycle. When the light phase was shifted by 5 and 17 h 1 week before the animals were born, sensitivity to melatonin action was unchanged. The responsiveness of the neuroendocrine-reproductive axis to exogenous melatonin was then studied throughout the day-night cycle. The inhibitory influence of melatonin increased gradually during the late photophase and reached a peak just before the onset of darkness. No effect was observed during the first 7 h of the dark phase; however, melatonin injected 9 h after the onset of darkness again had an inhibitory influence equivalent to about 50% of that observed in the late photophase , whereas administration 2 h later remained without effect. These results demonstrate that the time of administration of melatonin within the day-night cycle is critical for melatonin action.(ABSTRACT TRUNCATED AT 250 WORDS)