Pseudomonas sp. strain NCIB 9816-4 utilizes naphthalene dioxygenase (NDO), a multicomponent enzyme system, to initiate naphthalene degradation. The terminal component of NDO is an iron-sulfur protein (ISP NAP) with an α 2β 2 subunit composition. The structural genes encoding the α ( nahAc) and β ( nahAd) subunits were cloned separately and together into expression vectors where transcription is under the control of the T7 promoter. The recombinant plasmids were transformed into Escherichia coli JM109[pGP1-2] and the synthesis of ISP NAP and its α and β subunits was determined by SDS-PAGE. Low expression of nahAd was shown to be due to inefficient initiation of translation, but a sixfold increase in the amount of β subunit synthesized was achieved in a coupled translation system. Inclusion bodies were found in all recombinants. Increased levels of soluble active proteins were obtained when E. coli JM109(DE3), used as the host strain for recombinant plasmid, was grown at 25°C. ISP NAP from JM109(DE3)[pDTG121] was purified to homogeneity and shown to have the same properties as those determined for the enzyme purified from NCIB 9816-4. Active ISP NAP was also obtained by mixing cell extracts from separate strains that synthesized the α and β subunits. The availability of large amounts of purified ISP NAP and its α and β subunits will facilitate future studies on the mechanism of oxygen fixation by NDO.