In this study we tested the hypothesis that exogenous melatonin exerts its effects on the reproductive system of hamsters by directly or indirectly altering the endogenous rhythm of melatonin production and release. Melatonin was injected in male hamsters housed on LD 14:10 (lights 0600-2000 hr) either at 1200 or 1900 hr (15 micrograms in 0.1 ml ethanol:saline 1:10) daily for 12 weeks. Testicular regression occurred in all animals of the 1900-hr injection group, while melatonin injected at noon was without effect. A third group of animals received small implants of melatonin subcutaneously at 0, 4, and 8 weeks. Implants were 4 mm in length and contained a melatonin:beeswax mixture (1:25) drawn up into polyethylene tubing (2.2 mm i.d.). These implants release approximately 10-15 micrograms melatonin/day, and had no effect on testicular size, as these animals also remained on LD 14:10. After 12 weeks the animals of each group were sacrificed at 1- or 2-hr intervals around the clock. Pineals were saved and assayed for melatonin content. In each group the nocturnal rhythm of pineal melatonin was similar; peak melatonin levels were achieved 6 hr after lights out (0200 hr) and levels remained elevated for approximately 4 hr. These results exclude a mode of action of exogenous melatonin on the pineal melatonin rhythm as a basis for the testicular response to melatonin in hamsters. They also pose some interesting questions of feedback regulation by melatonin on its own production and release.