This study presents the first report of the purification and characterization of a monooxygenase enzyme from Pseudomonas putida A (ATCC 12633) that is responsible for the oxidation of physiologically relevant quaternary ammonium compounds, the tetradecyltrimethylammonium bromide. The degradation of tetradecyltrimethylammonium bromide by P. putida A (ATCC 12633) is initiated by N-dealkylation and catalysed by tetradecyltrimethylammonium monooxygenase (TTABMO), resulting in the formation of tetradecylalkanal and trimethylamine. Based on sequence analysis, the gene for TTABMO (ttbmo) corresponded to an ORF named PP2033 in the genome of P. putida KT2440. Mutation in ttabmo blocked the utilization of tetradecyltrimethylammonium bromide by Pseudomonas putida A (ATCC 12633) as carbon and nitrogen sources. The enzyme can be highly overexpressed in P. putida Δttabmo-T7 in active form and purified as a hexahistidine fusion protein. Like the native enzyme, the his-TTABMO was found to be a monomer with molecular mass of 40 kDa, the isoelectric point 7.3, that catalyses the breakdown of tetradecyltrimethylammonium bromide and utilized NADPH and FAD as cofactor. The biochemical properties and the analysis of the respective protein sequence revealed that TTABMO represents a typical flavoprotein monooxygenase, which is member of a flavoprotein family that is distinct from Baeyer-Villiger monooxygenases.