A phenol sulfotransferase from rat liver (EC 126.96.36.199), expressed in Escherichia coli from a single cDNA, was purified as two separable but catalytically active proteins. The proteins appeared to be identical to each other and to the natural liver sulfotransferase by comparison of their amino acid constitution, amino-terminal end group, and interaction with a polyclonal antibody raised against the liver enzyme. Each of the recombinant forms, α and β, catalyzed the sulfuryl group transfer from 4- nitrophenylsulfate to an acceptor phenol, a reaction in which 3'-phospho- adenosine 5'-phosphate (PAP) is a necessary intermediate. Only form β, however, catalyzes the physiological transfer of a sulfuryl group from 3'- phosphoadenosine 5'-phosphosulfate (PAPS) to the free phenol. Evidence is presented that sulfotransferase α, but not β, has 1 mol of PAP tightly bound per enzyme dimer. The ability to utilize PAPS as a sulfate donor could be altered: form could be treated and purified as form β to acquire the ability to use PAPS, whereas form β was treated by extended incubation with PAP, lost its ability to use PAPS, and was purified as form α.