A single s.c. injection of hCG (100 i.u.) produced a biphasic serum testosterone response in adult male rats, peaks being noted at 2 hr (24 ng/ml) and 3 days (16 ng/ml). The levels fell to control during the intervening interval (8 ng/ml), although there were elevated levels of serum hCG. Maintenance of high oestradiol levels by a s.c. injection of 50 μg oestradiol benzoate given on Day 2 after the initial hCG injection failed to prolong the refractory period and the secondary peak of testosterone (16 ng/ml) occurred on Day 3. Administration of the antioestrogen, tamoxifen (2 mg or 3 μg), 24 hr before or simultaneously with hCG did not prevent testicular refractoriness in vivo because serum testosterone levels still declined after 2 hr to reach a nadir at 2 days. The basal in-vitro testosterone production by decapsulated testes from animals injected with hCG was enhanced at 2 hr. Stimulation by hCG increased the amount of testosterone produced (x 1.5 that in controls). By 12 hr basal production decreased and there was no further increment in testosterone in the presence of hCG. This refractoriness to further hCG stimulation prevailed until Day 3, but the total production of testosterone fell so that at 24 hr and 2 days testes were producing basal amounts of testosterone. Testes recovered from refractoriness at 4 and 5 days, when basal and stimulated testosterone production were greater than in controls. Injection of 50 μg oestradiol benzoate at 2 days did not prolong the in-vitro refractory period and 2 mg or 3 μg tamoxifen had no effect on the in-vitro steroidogenic activity, since testes were still refractory to further hCG stimulation from 12 hr to 3 days. The results of the present study do not support the hypothesis that oestradiol is involved in the hCG-induced refractoriness of the Leydig cell. The nadir between the peaks of serum testosterone in vivo corresponds to the period during which the testis is refractory to in-vitro stimulation by hCG.