In a previous paper we demonstrated that properly timed 1-hr infusions of 50 ng melatonin effectively suppressed testicular development in juvenile Siberian hamsters, Only melatonin infused between 20:00 and 21:00 hr was effective in animals exposed to 16L (lights off 20:00 hr), In this paper we further investigate the importance of the coincidence and duration hypotheses of daily exposure of melatonin. Prepubertal Siberian hamsters received either 4- or 8-hr melatonin infusions at various times either on long photoperiod (LD 16:8 = 16L) or on short photoperiod (LD 10:14 = 10L). Daily 8-hr melatonin infusions suppressed testicular development in both photoperiods. Daily 4-hr, 50 ng/hr, melatonin infusions at 17:00-21:00 hr inhibited testicular growth in 16L and daily 4-hr melatonin infusions (either 50 ng/h or 50 ng/day) inhibited testicular growth at 17:00-21:00 hr in 10L. We also tested the efficacy of an interrupted melatonin infusion of long duration (8 hr), Pinealectomized prepubertal male Siberian hamsters, burn on 16L, were infused with two signals of 4 hr separated by an interval of 2 hr. Melatonin-infused groups had significantly inhibited testicular growth compared to vehicle-infused animals. Testicular development was maximally inhibited only in those groups in which the period of melatonin sensitivity identified in the previous paper (20:00-21:00 hr) overlapped or immediately followed a period of melatonin infusion. Considering the restrictions of the experimental design employed in these studies, the results are best explained by the hypothesis that the photoperiodic gonadal response in juvenile Siberian hamsters is regulated by the coincidence in time of exogenously administered melatonin with an intrinsic rhythm of sensitivity to melatonin, which occurred at 20:00-21:00 hr, The duration of the melatonin signal alone can not explain the results.