The cDNA for human liver phenol-sulfating phenol sulfotransferase (P-PST) has been cloned and the active enzyme expressed in Cos cells and bacteria. Analysis of the sequence identified two cysteine residues, one of which is highly conserved in the phenol sulfotransferase gene family. Previous studies with the pure human liver enzyme suggested that the conserved cysteine may be involved in binding substrates. Bacterial expression of P-PST with the cysteine converted to a serine indicates that the cysteine is not essential for activity or substrate binding, however, the mutant enzyme is significantly more sensitive to thermal inactivation.