Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Biginelli reactions have been monitored by direct infusion electrospray ionization mass spectrometry (ESI-MS) and key cationic intermediates involved in this three-component reaction have been intercepted and further characterized by tandem MS experiments (ESI-MS/MS). Density functional theory calculations were also used to investigate the feasibility of the major competing mechanisms proposed for the Biginelli reaction. The experimental and theoretical results were found to corroborate the iminium mechanism proposed by Folkers and Johnson, whereas no intermediates directly associated with either the more energy demanding Knoevenagel or enamine mechanisms could be intercepted.153897999804Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ)FINEPConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)