A more general approach to native (amide-forming) chemical ligation of unprotected peptide segments is described that extends the technique beyond the previously reported X-Cys ligation site to now include X-Gly and Gly-X ligation sites. A peptide, [peptide(1)](alpha)COSR, is reacted with a second peptide, HSCH2CH2(O)-N-alpha[peptide(2)], under conditions promoting thioester exchange. The intermediate thioester-linked product rearranges to form a ligation product linked by an N-substituted amide bond. If desired, the -oxyalkyl substitution on the amide bond can be removed by facile treatment with Zn in acidic medium to give a native peptide bond at the ligation site. The techniques described have been employed to ligate small model peptide segments to yield peptides with native or modified backbones, proving the feasibility of this approach.