Surface proteins of Staphylococcus aureus are anchored to the cell wall envelope by a mechanism requiring a C-terminal sorting signal with an LPXTG motif. Sortase A cleaves surface proteins between the three-nine (T) and the glycine (G) residues of the LPXTG motif and catalyzes the formation of an amide bond between the carboxyl group of threonine at the C-terminal end of polypeptides and the amino group of pentaglycine cross-bridges of cell wall peptidoglycan. Previous work showed that Cys184 and His120 of sortase A are absolutely essential for catalysis; however an active site thiolate-imidazolium ion pair may not be formed. The three-dimensional crystal structure of sortase A revealed that Arg197 is located in close proximity to both the active site Cys184 and the scissile peptide bond between threo-nine and glycine. We show here that substitution of Arg197 with alanine, lysine, or histidine severely reduced sortase A function both in vivo and in vitro, whereas Asn88, which had earlier been implicated in hydrogen bonding to His120, was found to be dispensable for catalysis. As the structural proximity of Arg197 and Cys 184 is conserved in sortase enzymes and as ionization of the Cys 184 sulfhydryl group seems required for sortase activity, we propose that Arg197 may function as a base, facilitating thiolate formation during sortase-mediated cleavage and transpeptidation reactions.