The role of the pituitary in the sexual differentiation of the Δ4-3-keto reduction of testosterone in th e rat liver was investigated. Testosterone-1,2-H3 was incubated in liver slices under nitrogen and the sexually dimorphic pattern of the metabolites 3α,17β-dihydroxy-5β-androstane (A), 3β, 17β-dihydroxy-5α-androstane (B), 3α, 17β-dihydroxy-5α-androstane (C) and 5α-dihydrotestos-terone (D) was studied. Male rats, gonadectomized at 25 days of age, formed as adults higher amounts of A and B and lower amounts of C and D than gonadectomized female rats. Hypophysectomy at 25 days of age abolished the development of these sex differences. Compared to gonadectomized females, hypophysectomized rats produced high levels of A and B and low amounts of C and D, which is a shift to a male-type of metabolism. Implantation of a male or female pituitary under the kidney capsule in gonadectomized-hypophysectomized rats did not restore sexual differentiation of metabolism but resulted in a decrease of A and B and a large increase of C and D. Irrespective of the sex of the donor, the pattern of metabolites obtained is typically feminine. The results suggest that sexual differentiation of liver testosterone metabolism requires the presence of the pituitary in situ and that it depends on a sex difference in pituitary hormone secretion, the regulation of which is probably located in the hypothalamus. (Endocrinology94: 1577, 1974)