In the Finnerty pathway for n-alkane oxidation in Acinetobacter sp,, n-alkanes are postulated to be attacked by a dioxygenase and the product, n-alkyl hydroperoxide, is further metabolized to the corresponding aldehyde via the peroxy acid [W. R. Finnerty, p. 184-188, in A. H. Applewhite (ed.), Proceedings of the World Conference on Biotechnology for the Fats and Oil Industry, 1988], However, no biochemical evidence regarding the first-step reaction is available, In this study, we found a novel n-alkane-oxidizing enzyme that requires only molecular oxygen, i.e., not NAD(P)H, in our isolate, Acinetobacter sp, strain M-1, and purified it to apparent homogeneity by gel electrophoresis. The purified enzyme is a homodimeric protein with a molecular mass of 134 kDa, contains 1 mol of flavin adenine dinucleotide per mol of subunit, and requires Cu2+ for its activity, The enzyme uses n-alkanes ranging in length from 10 to 30 carbon atoms and is also active toward n-alkenes (C-12 to C-20) and some aromatic compounds with substituted alkyl groups but not toward a branched alkane, alcohol, or aldehyde, Transient accumulation of n-alkyl hydroperoxide was detected in the course of the reaction, and no oxygen radical scavengers affected the enzyme activity, From these properties, the enzyme is most probably a dioxygenase that catalyzes the introduction of two atoms of oxygen to the substrate, leading to the formation of the corresponding n-alkyl hydroperoxide, The enzymatic evidence strongly supports the existence of an n-alkane oxidation pathway, which is initiated by a dioxygenase reaction, in Acinetobacter spp.