The goal of this study was to discriminate between two hypotheses regarding how the circadian rhythm of pineal melatonin (MEL) production transmits photoperiodic information: (1) A circadian rhythm of sensitivity to MEL regulates the hormone's effect; (2) the duration of the MEL signal, rather than its circadian timing, is the critical parameter of the MEL rhythm. The experiment examined the response of pinealectomized (PINX) male Siberian hamsters to 10-hr (short-day-type) versus 6-hr (long-day-type) duration MEL infusions (10 ng/infusion) in cycles with period lengths (T) of 18, 24, 36, and 48 hr. After cannula implantation, animals were moved from LD 16:8 to LD 10:14 (lights-on from 0500 to 1500 hr, EST), where the timed infusions began. Additional T 24 cycles included as controls employed 18-hr MEL, 18-hr saline (SAL), and 10-hr SAL infusions: Body weight and food intake were measured weekly. After 6 weeks, animals were killed; blood samples were taken for radioimmunoassay (RIA) of serum follicle-stimulating hormone (FSH) and prolactin (PRL); and terminal body, epididymal white adipose tissue (EPIWAT), and paired testis weights were recorded. Six-hour MEL infusions failed to induce short-day-type effects, regardless of the period (T) of the infusion cycle. In contrast, compared to SAL and 6-hr MEL infusions, 10-hr MEL resulted in decreases in body, EPIWAT, and testis weights in T 24, but not in T 36 or T 48. In T 18, testis, body, and EPIWAT mass were decreased, but not to the same extent as in T 24. Similarly, daily 18-hr MEL infusions (T24) were less effective as a short-day stimulus than were 10-hr MEL infusions. The effectiveness of 10-hr, but not 6-hr, MEL infusions in T 18 and T 24 is consistent with the duration hypothesis and argues against the circadian hypothesis. Neither hypothesis could have predicted that all infusion cycles of T greater than or equal to 36 hr, regardless of the infusion durations, would fail to elicit short-day-type responses. This outcome suggests a need for relatively frequent (T less than 36 hr) MEL stimulation in addition to the requirement for adequate duration of each MEL infusion.