Hepatic bile salt sulfotransferase activity was extremely limited in fetus, gradually increasing after birth. At puberty, enzyme activity declined in males but not in females, suggesting the influence of gonadal hormones associated with sexual maturation. Extremely high enzyme activity was found in pregnant rats at term. The neonatal bile salt sulfotransferase activity could be stimulated by bile acid feeding during pregnancy or maternal bile duct ligation. In contrast, a decrease in enzyme activity was detected in the treated pregnant females. Phenobarbital treatment during pregnancy also produced a 5-fold increase in neonatal enzyme activity. These results suggested that bile salt sulfation was regulated by chemical factors before maturity, and by gonadal hormones thereafter. Two fractions with bile salt sulfotransferase activity were separated from female liver by zone electrophoresis and DEAE-Sephadex A-50 chromatography, while a single active fraction was detected in male liver which corresponded to one of the active female fractions. The two active fractions in the female exhibited the same molecular weight (130 000), and different isoelectric points (6.8 and 5.3). The male fraction had a molecular weight of 130 000 and a pI of 5.3.