The mature female rat has three times the hepatic bile salt sulfotransferase (BSS) activity compared with male rats. This study examined the changes in two hepatic BSS isoenzyme activities during sexual maturation, and the role of estrogen in development of sex differences in BSS activities in mature rats. DEAE-Sephadex A-50 chromatography of hepatic cytosol from prepubescent pups revealed that more than 90% of total BSS activity was due to BSS I activity relative to BSS II, similar to postpubertal females. Sex differences in total BSS activities and the isoenzyme patterns developed after the onset of puberty at 30-35 days of age. BSS I was still the predominant isoenzyme in the adolescent female, similar to the prepubescent pup and mature female. In contrast, BSS I activity declined in adolescent males, which appeared to explain the fall in total BSS activity to only one-third of that of the female by maturity. BSS II activity was similar in both sexes at any age. Estrogen treatment of postpubertal male rats rapidly increased hepatic BSS capacity by enhancing BSS I activity producing an isoenzyme pattern similar to the mature female. This rapid enhancement of BSS I by estrogen was blocked by actinomycin D and puromycin. We concluded that 1) sex differences in BSS activities that develop during adolescence were in part due estrogen-maintaining BSS I activity in females and 2) estrogen regulates the synthesis of BSS I at a translational (or pretranslational) level.