A bacterium (strain B1) utilizing hexadecyltrimethylammonium chloride as a carbon and energy source was isolated from activated sludge and tentatively identified as a Pseudomonas sp. This bacterium only grew on alkyltrimethylammonium salts (C12 to C22) and possible intermediates of hexadecyltrimethylammonium chloride breakdown such as hexadecanoate and acetate. Pseudomonas strain B1 did not grow on amines. Simultaneous adaptation studies suggested that the bacterium oxidized only the alkyl chain of hexadecyltrimethylammonium chloride. This was confirmed by the stoichiometric formation of trimethylamine from hexadecyltrimethylammonium chloride. The initial hexadecyltrimethylammonium chloride oxygenase activity, measured by its ability to form trimethylamine, was NAD(P)H and O2 dependent. Finally, assays of aldehyde dehydrogenase, hexadecanoyl-coenzyme A dehydrogenase, and isocitrate lyase in cell extracts revealed the potential of Pseudomonas strain B1 to metabolize the alkyl chain via beta-oxidation.