The behaviour of some N,N′-disubstituted hexahydropyrimidines, a class of naturally occurring compounds of biological and biomedical interest, has been studied in both electron ionization (EI) and electrospray ionization (ESI) modes coupled with collisional experiments (ESI-MSn). In both techniques, the [M–H]+ ions are highly abundant, even if their formation is generated by two different mechanisms, i.e. H. loss from the M+. species in the case of EI and hydride (H−) abstraction from the molecules in the case of ESI. Furthermore, due to the low, step-by-step internal energy deposition typical of collisional experiments performed in an ion trap mass spectrometer, different fragment ions were observed in EI and ESI-MSn collisions. In both cases, the ions can be related to the original structure and allow us to identify the positions in which the different substituents are present. Copyright © 2006 John Wiley & Sons, Ltd.