A radioimmunoassay was developed for measuring testosterone (T) in the plasma of the rhesus monkey (Macaca mulatto). The method is sensitive and specific and can distinguish 20 pg of T when added to plasma from the adrenalectomized- ovariectomized monkey. When plasma obtained from cord blood at various times during gestation was analyzed by this method, the levels of T in plasma from the umbilical artery of males were significantly higher than those in females. Plasma from females did, however, contain small amounts of this hormone with little variation between animals. Large fluctuations in the concentrations of T in plasma from the fetal male were observed. Significantly higher amounts of T were found in the umbilical artery than in the vein in male but not in female fetuses. Castration of the fetus on day 100 of gestation abolished the sex difference in the amounts of T found on day ISO to 156 of gestation. These data indicate that the fetal testis of the monkey is the source of the elevated levels of T found in males and that fetal males develop in a different hormonal milieu than fetal females. (Endocrinology93: 156, 1973)