Four pairs of positional isomers of ureidopeptides, FmocNH-CH(R1)-φ(NH-CO-NH)-CH(R2)-OY and FmocNH-CH(R2)-φ(NH-CO-NH)-CH(R1)-OY (Fmoc = [(9-fluorenyl methyl)oxy]carbonyl; R1 = H, alkyl; R2 = alkyl, H and Y = CH3/H), have been characterized and differentiated by both positive and negative ion electrospray ionization (ESI) ion-trap tandem mass spectrometry (MS/MS). The major fragmentation noticed in MS/MS of all these compounds is due to NCH(R)Nbond cleavage to form the characteristic N- and C-terminus fragment ions. The protonated ureidopeptide acids derived from glycine at the N-terminus form protonated (9H-fluoren-9-yl)methyl carbamate ion at m/z 240 which is absent for the corresponding esters. Another interesting fragmentation noticed in ureidopeptides derived from glycine at the N-terminus is an unusual loss of 61 units from an intermediate fragment ion FmocNH = CH2+ (m/z 252). A mechanism involving an ion-neutral complex and a direct loss of NH3 and CO2 is proposed for this process. Whereas ureidopeptides derived from alanine, leucine and phenylalanine at the N-terminus eliminate CO2 followed by corresponding imine to form (9H-fluoren-9-yl)methyl cation (C14H11+) from FmocNH = CHR+. In addition, characteristic immonium ions are also observed. The deprotonated ureidopeptide acids dissociate differently from the protonated ureidopeptides. The [M − H]− ions of ureidopeptide acids undergo a McLafferty-type rearrangement followed by the loss of CO2 to form an abundant [M − H − Fmoc + H]− which is absent for protonated ureidopeptides. Thus, the present study provides information on mass spectral characterization of ureidopeptides and distinguishes the positional isomers. Copyright © 2010 John Wiley & Sons, Ltd.