These studies elucidate the ontogeny of two classes of estrogen-binding sites present in rat liver cytosol. The first class of sites is precipitated from whole cytosol fractions by ammonium sulfate (30% saturation) and exhibits characteristics of specific estrogen receptors. Detectable levels of receptors are attained during the third postnatal week. During days 30--40, receptors reach maximum concentration and remain relatively constant thereafter. The second class of sites, detected in whole cytosol fractions, possess a high binding capacity for estrogens and are present in similar amounts in male and female liver before day 34. However, between days 34--40 male levels increase dramatically while female levels remain constant. This sex difference is maintained throughout the duration of the study (160 days). Specific estrogen receptors from immature (26 days) and mature (70--80 days) rat liver have similar characteristics in terms of sedimentation properties in sucrose gradients, ligand binding specificity, and heat and pronase susceptibility. After prepuberal (19--21 days) gonadectomy, levels of receptor in subsequent adult animals of both sexes are increased approximately 40%. No alterations in receptor levels are seen after neonatal (day 1) castration of males. Prepuberal (day 19) gonadectomy does not alter the normal development of sexual differentiation of high capacity estrogen-binding sites. However, after neonatal (day 1) castration male rats, levels of those sites do not undergo sexual differentiation, and neonatally castrated adult males exhibit female levels of high capacity estrogen-binding site. These studies suggest that sexual differentiation of high capacity estrogen-binding sites may be programmed at birth by testicular androgens.