The effect of cone voltage (CV) variation on the mass spectral pattern of the bisquaternary ammonium salt decamethoxinum in the electrospray ionization (ESI) mode was studied. The advantage of decamethoxinum as a test compound in ESI mass spectrometry lies in the production of two types of precursor ions, i.e. the doubly charged organic dication Cat2+ and its singly charged cluster with a Cl− counterion, Cat · Cl+. This makes it possible to monitor the fragmentation patterns of these ions under identical experimental conditions. Pronounced qualitative and quantitative changes in the ESI mass spectra were observed upon a gradual increase of the CV. The model compound decamethoxinum allowed us to reveal the extreme situation, in which the mass spectra at a CV below and over approximately 100 V look quite different, in that they contain different product ions of Cat2+ and Cat · Cl+. While this effect may be much less pronounced for other classes of organic compounds, it should be properly taken into account for the adequate description of fragmentation pathways of bisquaternary ammonium compounds with ESI. Comparison of ESI, FAB-SIMS and MALDI mass spectra of decamethoxinum shows that taking into account CV effects also permits us to gain information on energy deposition into ions generated with the different ionization techniques. Copyright © 2006 John Wiley & Sons, Ltd.