Sulfotransferases (STs) catalyze all the known biological sulfonations, in which a sulfuryl group from a common sulfonate donor such as 3′-phosphoadenosine 5′-phosphosulfate (PAPS) is transferred to a nucleophilic acceptor. In addition to PAPS, phenol sulfotransferase (PST), a member of the ST family, utilizes other nucleotides as substrates with much less catalytic efficiency [Lin, E. S., and Yang, Y. S. (2000) Biochem. Biophys. Res. Commun. 271, 818-822]. Six amino acid residues of PST have been chosen for mutagenesis studies on the basis of a model of PST and its sequence alignment with those of available cytosolic and membrane-anchored STs. Systematic analyses of the mutants reveal that Ser134 is important for the regulation of nucleotide specificity between 3′-phosphoadenosine 5′-phosphate (PAP) and adenosine 5′-monophosphate (AMP). Kinetic studies also indicate that Ser134 plays a key role in nucleotide binding (Km) but not in catalysis (kcat). Consequently, the catalytic efficiency (kcat/Km) of PST can be altered by 5 orders of magnitude with a mutation of Ser134. Moreover, the change in nucleotide specificity from PAP to AMP can be achieved by mutation of Ser134 to any of the following residues: Glu, Gln, Arg, and His. Roles of Lys44, Arg126, and Arg253, which interact directly with the 5′- and 3′-phosphate of PAP, were also investigated by mutagenesis and kinetic experiments. On the basis of these findings, we suggest that Ser134 is the key residue that enables PST to discriminate PAP from AMP.