Melatonin disturbs the reproductive process in seasonal but not in continuous breeders: however, it delays sexual development in the rat. The testicular function of rats injected daily with melatonin from 20 up to 25, 30, 35 or 40 days of age was analyzed. The spermatogenic and androgenic activity of testes was assessed by light microscopy and by the capacity for binding hCG and producing testosterone in vitro, respectively. In addition, LH, FSH and testosterone plasma levels were measured and 3B-hydroxysteroid dehydrogenase activity of Leydig cells was assessed histochemically to aid in the interpretation of results. Rats treated with melatonin for 15 or 20 days presented at the end of the juvenile period, abnormal progression of spermatogenesis and decreased ability of their Leydig cells to produce testosterone both in vivo and in vitro. This was associated with a lower number of binding sites for hCG and diminished production of testosterone in response to receptor and post-receptor mediated stimulation of steroidogenesis. Melatonin caused a marked decrease in LH serum levels. The diminished LH supply to the testis probably interfered with differentiation or impaired the functional ability of Leydig cells. As a consequence, testicular testosterone production was insufficient to support normal spermatogenic progression and growth and development of the sexual accessory organs.