A radioimmunoassay for serum testosterone that does not require chromatographic purification of samples was developed and used to describe the temporal pattern of this steroid in adult male rhesus monkeys. Serum gonadotropins were measured by radioimmunoassays previously described. Testosterone levels in male rhesus monkeys show a threefold diurnal variation with peak levels of 17.0 ± 1.5 (SEM) ng/ml appearing at 2100 h. No concomitant variations in circulating LH are observed, however. Adrenal androgen secretion does not contribute significantly to this nocturnal testosterone elevation since castrated males show no circadian variation in testosterone levels. In orchidectomized animals implanted with Silastic capsules containing testosterone, the resulting serum levels of the steroid are constant, indicating that changes in metabolic clearance rate cannot account for the testosterone fluctuations seen in the intact male. These observations suggest that the diurnal shifts in testosterone concentration are the consequence of changes in testicular secretion rate. In contrast to intact animals, orchidectomized monkeys show a significant nocturnal elevation in serum LH concentrations suggesting that, in normal males, increases in circulating LH too small to be measured with our radioimmunoassay may account for the nocturnal rise in serum testosterone levels.