The traceless Staudinger ligation enables the formation of an amide bond between a phosphinothioester (or phosphinoester) and an azide without the incorporation of residual atoms. Here, the coupling of peptides by this reaction was characterized in detail. Experiments with [(18)O]H(2)O indicated that the reaction mediated by (diphenylphosphino)methanethiol proceeded by S-->N acyl transfer of the iminophosphorane intermediate to form an amidophosphonium salt, rather than by an aza-Wittig reaction and subsequent hydrolysis of the resulting thioimidate. A continuous (13)C NMR-based assay revealed that the rate-determining step in the Staudinger ligation of glycyl residues mediated by (diphenylphosphino)methanethiol was the formation of the initial phosphazide intermediate. Less efficacious coupling reagents and reaction conditions led to the accumulation of an amine byproduct (which resulted from a Staudinger reduction) or phosphonamide byproduct (which resulted from an aza-Wittig reaction). The Staudinger ligation mediated by (diphenylphosphino)methanethiol proceeded with a second-order rate constant (7.7 x 10(-3) M(-1) s(-1)) and yield (95%) that was unchanged by the addition of exogenous nucleophiles. Ligations mediated by phosphinoalcohols had lower rate constants or less chemoselectivity. Accordingly, (diphenylphosphino)methanethiol was judged to be the most efficacious known reagent for effecting the traceless Staudinger ligation.