Oligosaccharyl transferase activity exhibits an absolute requirement for certain divalent metal cations. Studies with reconstituted enzyme suggest a preference for metal ions that can adopt an octahedral coordination geometry. In order to gain insight into the specific role of the metal cation in catalysis, we have investigated the influence of the metal cofactor on catalytic turnover of the tripeptide substrate Bz-Asn-Leu-Thr-NHMe (1) and a closely related sulfur-containing analog, Bz-Asn(gamma S)-Leu-Thr-NHMe (2). The metal ion substitution studies reveal that 1 is effectively turned over in the presence of several metal ions (Mn2+, Fe2+, Mg2+, and Ca2+). In contrast, 2 is only glycosylated in the presence of the thiophilic metal cations manganese and iron. When the enzyme is reconstituted with the oxophilic cations magnesium and calcium, 2 shows minimal substrate behavior. With the amide substrate 1, the distinct preference for manganese over magnesium may argue against direct coordination of the metal to the lipid-linked substrate pyrophosphate moiety. This fact, together with the comparative studies with asparagine- and thioasparagine-containing tripeptides, implicates the metal cofactor in a role that places it proximal to the peptide binding site.