Many of the sex-differentiated functions of the liver of adult male rats depend upon the occurrence of neonatal androgen imprinting, a brief surge of androgen early in life. We investigated whether androgen imprinting is necessary for the development and maintenance of levels of a microsomal enzyme, estrogen-2-hydroxylase (E-20Hase), and a male-specific cytosolic estrogen binder (MEB) which are higher in adult male than in adult female rats. Cimetidine, a weakly antiandrogenic H2 blocker, was administered to pregnant and lactating rats from day 12 of gestation through weaning on day 21. Livers of male progeny, 120-150 days of age, were assayed for E-2OHase and MEB activity; a maternal dose equivalent to 2.5 times the usual human dose inhibited MEB activity in the levels of the offspring by 40% but had no effect on E-2OHase. However, a higher dose (5 times the human dose) was effective in reducing the E-2OHase activity by 50%. Rats whose mothers had received either no drug or an equivalent dose of ranitidine, another H2 blocker without antiandrogenic activity, were used as controls. The groups were not different in hepatic cytosolic androgen receptor content, body weight, or serum testosterone. In other studies, the requirement of neonatal androgen imprinting for full expression of adult levels of MEB and E-2OHase was determined. Female rats, which have low levels of E-2OHase and undetectable levels of MEB, were given androgen on day 1, on day 60 after ovariectomy, or at both times. Levels of E-2OHase equivalent to those in adult males were induced in females receiving both androgen treatments, whereas either treatment alone induced E-2OHase to the level of that in males castrated at adulthood or neonatally, which is about 50% that in normal male controls. In addition, MEB levels were induced in females to 85% of that in normal males by both androgen treatments and to 50% by administration of androgen to adult females or to adult males castrated neonatally. Administration of androgen to females during the neonatal period only did not induce MEB. We conclude that both MEB and E-2OHase require androgen imprinting for full expression in adult male rats.