We report here the development of a robust recombinant expression system for Drosophila melanogaster tyramine beta-monooxygenase (TOM), the insect analog of mammalian dopamine beta-monooxygenase. Recombinant TOM is rapidly purified from the host cell media in three chromatographic steps. The expression system produces similar to 3-10 mg of highly purified, active protein per liter of culture. Recombinant T PM requires copper for activity and has a typical type 2 copper EPR spectrum. While TOM efficiently hydroxylates the aliphatic carbon of phenolic amines such as tyramine (the physiological substrate) and dopamine, phenethylamine is a poor substrate. TOM is most likely a monomer under physiological conditions, although under conditions of high pH and low ionic strength the dimeric form predominates. The lower oligomeric state of TOM may provide an advantage for structural studies over D beta M, which exists as a mixture of dimer and tetramer. (c) 2005 Elsevier Inc. All rights reserved.