The microheterogeneous peptaibol alamethicin F30 isolated from the culture broth of Trichoderma viride was analyzed by nonaqueous CE-electrospray-MS using an IT and a TOF mass analyzer. Compared to aqueous buffers, higher separation selectivity was observed for methanolic BGE allowing the detection of more minor components. The low electrophoretic mobility observed for neutral analytes under nonaqueous conditions may be explained by ion-dipole interactions between the peptide analytes and electrolyte ions. The amino acid sequences of the individual components were derived from MSn using the doubly or triply charged pseudomolecular ions as well as characteristic fragments as precursor ions. The exchange of Ala by a-aminoisobutyric acid (Aib) which is frequently observed for peptaibols was detected for several components. Additional variations included the exchange of Gln to Glu, and the loss of the C-terminal amino alcohol or of the first six amino acids from the N-terminus with concomitant formation of pyroglutamyl residues. In most cases comigration of the Aib peptaibols with the respective Ala component was observed as the mass difference of 14 Da as the result of the amino acid exchange was not sufficient to translate into an electrophoretic separation under the conditions applied. However, proper selection of the precursor ions allowed the unequivocal analysis of the components. Additional TOF-MS measurements were performed in order to resolve the ammonium adducts from comigrating compounds (i.e., Aib-Ala exchange) and to confirm the amino acid composition of the individual components. Except for neutral compounds migrating close to the EOF the mass accuracy was better than 4 ppm for the doubly charged pseudomolecular ions and better than 2 ppm for triply charged ions.