The technique of native chemical ligation has enabled the total chemical synthesis of proteins with molecular weights far in excess of those achievable by conventional stepwise solid-phase peptide synthesis. The method involves the condensation of two unprotected peptide segments, one bearing a C-terminal (alpha)thioester and the other an N-terminal cysteine residue, to afford a protein with a native amide linkage at the site of ligation. Here we report an extension of the native chemical ligation method to the total synthesis of a glycosylated protein, the antimicrobial O-linked glycoprotein diptericin. The major challenge in our synthesis was preparation of a 24-residue glycopeptide-(alpha)thioester segment, which was complicated by the incompatibility of glycosidic Linkages with Boc chemistry and by the incompatibility of thioesters with Fmoc chemistry. The use of an alkanesulfonamide " safety-catch" linker circumvented this problem and permitted the solid-phase synthesis of the glycopeptide-athioester using standard Fmoc chemistry protocols. Ligation of this thioester with a 58-residue glycopeptide bearing an N-terminal cysteine residue yielded the full-length glycoprotein with two sites of glycosylation. The fully deprotected diptericin glycoform was active in antimicrobial assays.