The ion formation mechanism in electrospray MS is reviewed, with special focus on the electrochemical red/ox reactions responsible for the formation of radical molecular ions. Prerequisites influencing the likelihood of formation and observation of a particular compound as an open-shell molecular species in ESI-MS (i.e., the structure and the oxidation potential of the analyte, the solvent and additives) are evaluated. For illustration of the ESI phenomena governing radical cation formation, an ESI-MS study of tetra(aryl)benzidine compounds is presented. The facile formation of abundant radical molecular cations in ESI-MS demonstrates imposingly that the basicity of the analyte's nitrogen atoms is strongly overcompensated by the ability to stabilize unpaired electrons. ESI-MSn spectra of the tetra(aryl)benzidine molecular ions exhibit a characteristic feature in the loss of radicals. This process is the major fragmentation pathway of open-shell molecular precursor ions in their MS2 spectra, and also that of even-electron ions in sequential MSn spectra. The collision-induced dissociation (CID) behaviour suggests a general assumption: easily oxidizable compounds (e.g., hydrocarbon polyenes, polycyclic aromatic hydrocarbons, porphyrins etc.) generating predominantly molecular radical cations in ESI-MS contradict the even electron rule in ESI-MSn experiments. The strong ability to stabilize unpaired electrons is preserved in product ions and makes the formation of open-shell species energetically less demanding. A selection of solution-phase reaction mechanistic studies in which open-shell intermediates were detected and structurally characterized by ESI-MS and ESI-MS/MS, respectively, is presented. The merits of ESI-MS and ESI-MSn for mechanistic studies of chemical reaction are critically discussed.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007)