A biphasic pattern of testosterone secretion in response to a single injection of 100 IU hCG has been observed in the rat. Serum testosterone increased from basal levels of 8.7 +/- 3.1 ng/ml (mean +/- SEM) to 23.0 +/- 1.4 ng/ml within 2 h of hCG-stimulation and returned to control levels by 2 days. A second, delayed, but significant increase in serum testosterone occurred, reaching a peak of 24.6 +/- 4.0 ng/ml at 3 days and declining to basal values at 5 days. To study this response further, lower doses of hCG were tried. Administration of 10 IU hCG produced a single peak of testosterone, which did not occur until 24 h. Differences in the serum testosterone response were related to the concentration of hCG measured in the serum after injection, as injection of 1 IU, which failed to increase serum hCG levels above detection, was also inadequate to increase serum testosterone. The response after stimulation with 500 micrograms ovine-LH or 0.1-10.0 micrograms LHRH was also evaluated. Injection of 500 micrograms ovine-LH produced a significant rise in serum testosterone reaching a peak at 2 h of 25.2 +/- 2.6 ng/ml and subsequently declining over the next 48 h to control levels where it remained for 5 days. Stimulation with doses of 0.1 - 10.0 micrograms LHRH produced rapid and short increase in serum LH concentration which induced peaks of testosterone up to 48.8 +/- 14.1 ng/ml 1 h post injection. No secondary peak of testosterone followed. Failure of ovine-LH and LHRH to produce a second testosterone peak suggests that this response may be due to a re-stimulation of the Leydig cell by elevated levels of hCG which persist until the fourth day after injection.