SunS is a novel S-glycosyltransferase involved in the biosynthesis of the antimicrobial peptide sublancin. It selectively modifies Cys22 in a 56 amino acid peptide substrate SunA and can accept a variety of NDP sugars. This study reports the substrate selectivity with regard to the peptide substrate and the antimicrobial activity of the resulting sublancin analogues. The results suggest that SunS recognizes an a-helix N-terminal of the Cys to be glycosylated, which is present in a flexible linker. Interestingly, when Cys22 is mutated, sugar attachment is not required for sublancin antimicrobial activity. Furthermore, the sublancin-producing strain Bacillus subtilis 168 also becomes susceptible to such mutants. These data suggest that S-glycosylation may be important for self-resistance.