A phenol sulfotransferase (3-phosphoadenylylsulfate:phenol sulfotransferase, EC 2.8.2.1) has been purified from rat liver using an affinity chromatography system consisting of a p-hydroxyphenylacetic acid-agarose conjugate. The ligand was separated from the insoluble matrix using a spacer of approx. 25 A degrees. When this affinity system was used in conjunction with other chromatography procedures, e.g., DEAE-cellulose and Sephacryl S-200, a 630-fold purification of the enzyme was achieved. The enzyme had a molecular weight of 69 000 as determined by gel filtration and 70 000 as determined by SDS-polyacrylamide gel electrophoresis. The enzyme readily sulfates p-nitrophenol, 2-naphthol, 1-naphthol, and salicylamide, as well as naturally occurring catecholamines. Using p-nitrophenol as the substrate, the pH optimum was determined to be in the range of 5.5 to 6.4. The Km value determined for p-nitrophenol was 3.6 microM, and that for 3'-phosphoadenosine 5'-phosphosulfate was 2.5 microM. Adenosine 3',5'-bisphosphate was found to be a strong product inhibitor with Ki = 0.4 microM.