We studied the regulation of hepatic bile salt sulfotransferase activity by gonadal hormones and the effect of gonadal hormones on two bile salt sulfotransferase isoenzymes. Bile salt sulfotransferase enzyme activity was three times greater in the female than in the male rats. Oophorectomy significantly decreased bile salt sulfotransferase activity in the female, but orchidectomy had no effect on bile salt sulfotransferase activity in the male. Estrogen treatment of intact as well as orchidectomized males markedly stimulated the enzyme activity, while testosterone treatment of intact or oophorectomized females did not effect bile salt sulfotransferase activity. We concluded that the 3-fold greater activity in female rats is due to the striking stimulatory effect of estrogen on bile salt sulfotransferase activity, and the testosterone has little or no role in the sexually related differences in bile salt sulfotransferase activity in mature rats. These sex-related differences in bile salt sulfotransferase activity were investigated further using DEAE-Sephadex A50 ion-exchange chromatography of rat hepatic cytosol. Two bile salt sulfotransferase isoenzymes were identified both with an approximate molecular weight of 130,000. Bile salt sulfotransferase I eluted with 0.05 M NaCl, had an isoelectric point at pH 6.8, was stimulated by estrogen, and was responsible for 90% of total bile salt sulfotransferase activity in the mature female. Bile salt sulfotransferase I1 eluted with 0.14 MNaCl, had an isoelectric point at pH 5.3, was unresponsive to estrogen, and accounted for 75 to 80% of bile salt sulfotransferase activity in the mature male. Bile salt sulfotransferase I was 3.8-fold more active toward 38-hydroxy-5-cholenoate as glycolithocholate, and its activity for glycolithocholate was completely inhibited by 3-ketolithocholate. Conversely, bile salt sulfotransferase I1 had very little detectable activity towards 3B-hydroxy-5cholenoate, and its activity toward glycolithocholate was only marginally (13%) inhibited by 3ketolithocholate. The activity of both bile salt sulfotransferase isoenzymes with various bile acids was: monohydroxylated > dihydroxylated > trihydroxylated. Hepatic bile salt sulfation in the mature rat is catalyzed by two isoenzymes that had the same approximate molecular weight, but differ in sex distribution, substrate activities, regulation by gonadal hormones and inhibition of activity for glycolithocholate by keto bile acids.