Abstract Electrospray ionization coupled with tandem mass spectrometry (MS/MS) was used to determine the preferred binding site(s) of biotin NHS ester with a series of cyclic peptides with antibiotic properties. The peptides investigated are polymyxins, cyclic peptides produced by Bacillus polymyxa . In spite of the 1:1 stoichiometry used in the labeling reaction, multiple biotin molecules were incorporated into intact polymyxin peptides. Given the amine specificity of the activated biotin and the large number of amino acids with primary amines in the polymyxins, it was not clear by inspection which binding sites were more reactive than others. MS/MS was used to characterize the structure of the biotinylated peptides. MS/MS spectra of cyclic peptides often lead to ambiguous structure determinations due to the potential for multiple ring openings which result in the generation of multiple ion series. The MS/MS spectra of polymyxin peptides are especially difficult to characterize due to the lack of variety in their amino acids; however, the added complexity of the biotin aided the elucidation of the fragmentation pathways. MS/MS spectra of the species with biotin additions were used to rationalize the preferential binding sites of these molecules. Published in 2004 by John Wiley & Sons, Ltd.