The stimulatory and inhibitory role of serotonin in gonadotropin secretion and in the onset of puberty in the male rat has been previously described, but its role in the establishment of spermatogenesis is not known. The aim of this study was to investigate the effects of serotoninergic inhibition by p-chloroamphetamine (pCA) on the prepubertal-to-adult stage of the rat reproductive system. Hypothalamic serotonin, gonadotropins and sex steroid hormone concentrations were measured, and a histopathological analysis of seminiferous epithelium was carried out on animals treated with pCA from day 30 and killed at 45 or 65 days of age. The pCA treatment significantly reduced the hypothalamic levels of serotonin and its metabolite (5-hydroxyindole-3-acetic acid). This inhibition did not affect the sex steroid hormone or LH concentrations, but rather it induced an increase in FSH concentration in animals of both ages. Spermatogenesis was impaired by pCA treatment. Disruption of seminiferous epithelium and the death of numerous germ cells were observed. Sperm produced by pCA-treated animals was of poor quality and appeared in small quantities. Apparently, serotonin depletion did not affect communication between the hypothalamus and the pituitary, but the FSH increase could have been related to alterations in the seminiferous epithelium effects. The seminiferous epithelium cycle was altered in rats killed at both 45 and 65 days of age, because at each age of killing the distribution of spermatogenesis stages was different. Germ cell apoptosis did not appear to be related to changes in the FSH concentrations, but other factors produced during spermatogenesis could have been involved in this induction. This study showed that serotonin was necessary for the development of normal spermatogenesis in prepubertal rats.