Steroid alcohol sulphotransferases (EC 2.8.2.—) have not previously been obtained in a pure state possibly because of their inherent instability. A rapid isolation procedure involving affinity chromatography was developed and initially applied to the isolation of dehydroepiandrosterone sulphotransferase from human adrenals, since the sulphate ester of this steroid is quantitatively (along with unconjugated cortisol) the most important secretory product of the human adrenal cortex. By use of the coupled product of dehydroepiandrosterone-17-( O-carboxymethyl) oxime and AH Sepharose 4B, the enzyme was isolated in one step from an (NH 4) 2SO 4 cut derived from the cytosol of human adrenal glands. Enzyme activity, employing dehydroepiandrosterone or etiocholanolone as substrates, was associated with the major band revealed on acrylamide gel electrophoresis. One or two very minor bands, lacking enzyme activity, were also usually present. On sodium dodecyl sulphate gels, a band having a molecular weight of 34 500 was obtained. By sucrose gradient ultracentrifugation the active enzyme was found to have a molecular weight of 68 000 and thus contains two subunits. These appear to be identical, as judged by fingerprint data of the tryptic peptides and the amino acid composition. Steroids other than dehydroepiandrosterone acted as substrates. The decreasing order of sulphurylation rates were: epiandrosterone, 1.6; androst-5-ene-3β,17α-diol, 1,4; dehydroepiandrosterone and pregnenolone, 1.0; etiocholanolone, 0.89; androst-5-ene-3β,17β-diol, 0.75; androsterone, 0.44; testosterone, 0.15; estradiol-17β, 0.17; 11-deoxycorticosterone, 0.10. Complex wave-like curves were obtained when either substrate, i.e. dehydroepiandrosterone or 3′-phosphoadenosine-5′-phosphosulphate, was varied in the presence of a fixed concentration of the cosubstrate. In marked contrast, enzyme isolated from bovine liver using the same affinity gel, yielded normal Michaelis-Menten kinetics.