Fatty aldehyde dehydrogenase (FALDH) is an NAD+-dependent oxidoreductase involved in the metabolism of long-chain fatty alcohols. Enzyme activity has been implicated in the pathology of diabetes and cancer. Mutations in the human gene inactivate the enzyme and cause accumulation of fatty alcohols in Sjögren-Larsson syndrome, a neurological disorder resulting in physical and mental handicaps. The major form of FALDH from human microsomes was expressed in E. coli and purified by anion-exchange and metal-chelate chromatographies. Using an in vitro activity spectrophotometric assay based on NAD+ reduction an optimum pH of ~9.5 and temperature of ~35 degrees C were determined. Medium- and long-chain fatty aldehydes were converted to the corresponding acids and kinetic parameters determined. The enzyme showed high activity with heptanal (227 ± 7 nmol/min/mg), tetradecanal (183 ± 19 nmol/min/mg), hexadecanal (195 ± 25 nmol/min/mg) and octadecanal (180 ± 25 nmol/min/mg) with lower activities for the other tested substrates. The enzyme was also able to convert some fatty alcohol substrates to their corresponding aldehydes and acids, with decanol at 44 ± 3 nmol/min/mg and hexadecanol at 64 ± 3 nmol/min/mg. A structural model of FALDH has been constructed, and catalytically important residues have been proposed to be involved in alcohol and aldehyde oxidation: Gln-120, Glu-207, Cys-241, Phe-333, Tyr-410 and His-411. These results place FALDH in a central role in the fatty alcohol/acid interconversion cycle, and provide a direct link between enzyme inactivation and disease pathology caused by accumulation of alcohols.