Hypophyseal gonadotropins (LH, FSH, and prolactin), as measured by radioimmunoassay, were compared in the sera and pituitaries of normal male and androgenized (AF) female rats; AF received 50 µg testosterone propionate at 5 days of age. In addition, the effect on these hormones of various doses (0.05–50 µg) of estradiol benzoate (EB) given for 4 days was evaluated. Serum concentrations of LH were not significantly different in AF and males and were unchanged by any dose of EB; sex accessory organ weights in the males were reduced, however. Pituitary LH concentration, which was much higher in males than in AF, was reduced by 0.5 µg EB, but total content did not change in either type of animal. Serum FSH was higher in males than in AF, and was reduced in both by 5 µg of EB; however, even with 50 µg of EB, the FSH in the male was about the same as that of normal diestrous females or adult AF. Pituitary concentration of FSH was 10 times greater in males than in AF. While a daily dose of 0.5 µgEB reduced this parameter in males, even the highest dose was without effect in AF. Prolactin was present in the serum of AF in very high concentration (147 ng/ml). The value was augmented by 30% following administration of 5 µg EB; but in the groups treated with 10 or 50 µg, the level was not different from that in oil-treated controls, i.e., estrogen did not inhibit prolactin output in these females. A slight increase in serum prolactin was noted in males receiving 0.5 µg EB, while a large increase was noted with 5 µg; higher doses did not change the concentration further. Pituitary prolactin content, which was lower in males than in AF, was unaffected by the estrogen treatment in both types of animals.