Sulfotransferases (STs)active on androsterone (AD), cortisol (CS) and 4-nitrophenol (NP) were separated by diethylaminoethyl-cellulose chromatography from cytosolic fractions of female rat liver and were divided into five ST fractions (peaks I-V) with different activities toward three substrates. The precipitates obtained in the 68% of saturation of ammonium sulfate were passed through a Sephadex G-100 column and purified by agarose-hexane adenosine 3', 5'-bisphosphate affinity chromatography. AD-ST isoenzyme (peak I) was purified 85-fold, had low CS-ST activity, was devoid of NP-ST activity and appeared to correspond to hydroxysteroid ST 1.Peaks II and V appeared to consist mainly of hydroxysteroid ST and arvl ST, respectively.