The comparison of the deduced amino acid sequences of plant and animal sulfotransferases (ST) has allowed the identification of four well conserved regions, and previous experimental evidence suggested that regions I and IV might be involved in the binding of the cosubstrate, 3'-phosphoadenosine 5'-phosphosulfate (PAPS). Moreover, region IV is homologous to the glycine-rich phosphate binding loop (P-loop) motif known to be involved in nucleotide phosphate binding in several protein families. In this study, the function of amino acid residues within these two regions was investigated by site-directed mutagenesis of the plant flavonol 3-ST. In region I, our results identify Lys59 as critical for catalysis, since replacement of this residue with alanine resulted in a 300-fold decrease in specific activity, while a 15-fold reduction was observed after the conservative replacement with arginine. Photoaffinity labeling of K59R and K59A with [35S]PAPS revealed that Lys59 is not required for cosubstrate binding. However, the K59A mutant had a reduced affinity for 3'-phosphoadenosine 5'-phosphate (PAP)-agarose, suggesting that Lys59 may participate in the stabilization of an intermediate during the reaction. In region IV, all substitutions of Arg276 resulted in a marked decrease in specific activity. Conservative and unconservative replacements of Arg276 resulted in weak photoaffinity labeling with [35S]PAPS and the R276A/T73A and R276E enzymes displayed reduced affinities for PAP-agarose, suggesting that the Arg276 side chain is required to bind the cosubstrate. The analysis of the kinetic constants of mutant enzymes at residues Lys277, Gly281, and Lys284 allowed to confirm that region IV is involved in cosubstrate binding.