Dissociation pathways of a series of alkali-cationized hybrid peptides, viz., Boc-α,β- and -β,α-carbopeptides, synthesized from C-linked carbo-β3-amino acids [Caa (S)] and α-alanine (L-Ala), have been investigated by electrospray ionization tandem mass spectrometry. The positional isomers (six pairs) of the cationized α,β- and β,α-peptides can be differentiated by the collision-induced dissociation (CID) spectra of their [M + Cat-Boc + H]+ ions which give characteristic series of alkali-cationized C- (x, y, z) and N-terminal (a, b, c) ions. Another noteworthy difference is cationized β,α-peptides eliminate a molecule of ammonia whereas this pathway is absent for α,β-peptides. This is useful for identifying the presence of a β-amino acid at the N-terminus. The CID spectra of [M + Cat-Boc + H]+ ions of these peptide acids show abundant rearrangement [bn + 17 + Cat]+ (n = 1 to n–1) ions which is diagnostic for distinguishing between α- and β-amino acid at the C-terminus. MSn experiments of [bn + Li–H]+ ions from these hybrid peptides showed the loss of CO and 72 u giving rise to [an + Li–H]+ and cationized nitrile product ions which render support to earlier proposals that b or [bn + Cat–H]+ ions have protonated or cationized oxazolinone structures, respectively. Copyright © 2006 John Wiley & Sons, Ltd.