This paper describes an orthogonal and stereospecific method for ligating free peptide segments to form a monosubstituted pseudoproline bond with a hydroxymethyl moiety at the C2 carbon. The pseudoproline ligation, comprising both the oxaproline and thiaproline ligations, initially involves an imine capture of a peptidyl glycoaldehyde ester with an N-terminal cysteine, serine, or threonine peptide segment and then two spontaneous cyclization reactions. The thiazolidine or oxazolidine ester formed in the first cyclization undergoes an O,N-acyl transfer to form an pseudoproline bond. The thiaproline ligation can be carried out exclusively with unprotected peptide segments in both aqueous and nonaqueous pyridine-acetic acid conditions. However, the oxaproline ligation is best performed in a nonaqueous pyridine-acetic acid mixture with unprotected peptide segments except for those containing N-terminal nucleophilic amino acids such as Cys, His, and Trp. Pseudoproline ligation is not only regioselective but also stereospecific. 2D H-1 NMR studies of dipeptide models, Z-Xaa-psi Pro-OMe, indicate that the newly created C2 stereocenter of the pseudoproline ring affords only an li-epimer and the C2-hydroxymethyl-substituted pseudoproline exhibits high preference for cis conformation. Three of the model peptides have more than 50% cis isomers. Finally, this novel method has been used successfully in ligating two segments of 24 and 35 amino acids under mild conditions to synthesize three analogues of bactenecin 7, an antimicrobial peptide containing 59 amino acid residues.