A wide diversity of biological molecules are modified by the addition of sugar residues, and a large number of glycosyltransferases have been identified that are responsible for these reactions. Despite catalyzing closely related reactions, many of these transferases show little apparent sequence homology. By comparing two apparently unrelated families of yeast Golgi mannosyltransferases, a short motif containing two aspartate residues was observed that was conserved in both groups of proteins. Mutagenesis of one of the members of these families, the alpha-1, 3-mannosyltransferase Mnn1p, showed that altering either of these aspartates eliminates all enzymatic activity. These changes do not appear to affect the overall folding and assembly of Mnn1p. A similar aspartate-containing sequence was found to be conserved in a diverse range of other glycosyltransferase families, much more frequently than would be expected by chance, suggesting that it is a feature of the catalytic site, or an element of a structural fold, shared by many glycosyltransferases.