Cytochrome P-450 from male and female rate liver microsomes has been solubilized with sodium deoxycholate, precipitated with ammonium sulphate and separated in the presence of deoxycholate into ten different fractions on a DEAE-cellulose column eluted with a stepwise gradient of KCL. Each fraction was characterized with respect to its ability to catalyze different hydroxylation reactions of free and disulphurylated 5alpha-androstane-3alpha,17 beta-diol, using a reconstituted system. All hydroxylases active on 5alpha-androstane-3alpha, 17 beta-diaol (i.e. the 2alpha-, 2beta-, 7alpha-, 7beta-, 12beta-, 15alpha-, 16alpha- and 18-hydroxylases) and 5alpha-androstane-3alpha, 17beta-diol 3,17-disulphate (i.e. the 15beta-hydroxylase) were solubilized with cholate. However, the 2beta- and 18-hydroxylases were partially inactivated when cholate was added to intact microsomes and these were also the only hydroxylase activities that could not be detected in reconstitution experiments with lipid, NADPH-cytochrome P-450 reductase and the cytochrome P-450 fractions recovered after DEAE-cellulose chromatography. Using microsomal preparations from male rat liver it was possible to obtain a partial separation of the 2alpha- and 7alpha-hydroxylases from the 12beta-, 15alpha- and 16 alpha-hydroxylases and also from the 7beta-hydroxylase. With preparations from female rat livers the l5beta was well separated from the 2alpha- and 7alpha-hydroxylases. The specific activities of the partially separated 2alpha-, 7alpha-, 7beta-, 12beta-, 15alpha- and 16alpha-hydroxylases calculated per nmol of cytochrome P-450 was 9--34% of the original activities in sonicated microsomes. There was an absolute requirement of the cytochrome P-450 fractions for all hydroxylase activities except for the 2alpha-hydroxylase activity. The requirement for NADPH-cytochrome P-450 reductase was not absolute for any of the hydroxylase activities and no lipid dependency was observed. Based on their behavior during solubilization and purification, elution pattern of the DEAE-cellulose column and different modes of regulation, the various hydroxylases studied can be divided into different groups. It is suggested that one form of cytochrome P-450 catalyzes the hydroxylation of 5alpha-androstane-3alpha,17beta-diol in the 2beta- and 18-positions, another form the 12beta-, 15alpha- and 16alpha-hydroxylations of the same substrate and a third form the 15beta-hydroxylation of 5alpha-androstane-3alpha,17beta-diol3,17-disulphate. It is concluded that rat liver microsomes contain multiple forms of cytochrome P-450 active on steroid hormones. It is suggested that the specificity of these forms is determined by the nature of the substrate binding site and by the distance between the binding and catalytic sites on the enzyme.