A gene (PP2216) that codes for an acyl-CoA dehydrogenase was cloned from Pseudomonas putida strain KT2240 and over-expressed in Escherichia coli, and the recombinant enzyme purified and characterised. The enzyme is tetrameric with one FAD per subunit of molecular mass 40,500 Da. An anaerobic titration with sodium dithionite showed that the enzyme accepts two electrons. A similar titration with butyryl-CoA showed that reduction by this substrate was incomplete with 4.5 mol butyryl-CoA added per mol enzyme FAD; the equilibrium was used to calculate that the oxidation-reduction potential of the enzyme at pH 7 and 25 degrees C is 5+/-5 mV versus the standard hydrogen electrode. The enzyme shows catalytic activity with butyryl-CoA, valeryl-CoA and hexanoyl-CoA, and very low activity with heptanoyl-CoA and octanoyl-CoA; it fails to oxidise propionyl-CoA. These properties resemble those of short-chain acyl-CoA dehydrogenases from other sources. The enzyme is inactive with the CoA derivatives of all phenylalkanoates that were tested (side chains 3-8 carbon atoms) indicating that in contrast to an earlier suggestion, the enzyme is not involved in the beta-oxidation of aromatic compounds.