Tetracenomycin (Tcm) F1 monooxygenase, which catalyzes the oxidation of the naphthacenone Tcm F1 to the 5,12-naphthacenequinone Tcm D3 in the biosynthesis of the anthracycline antibiotic Tcm C in Streptomyces glaucescens, has been purified to homogeneity and characterized. Gel filtration chromatography yields a molecular weight of 37,500 whereas SDS-PAGE gives a single band with a molecular weight of 12,500, indicating that the Tcm F1 monooxygenase is a homotrimer in solution. The N-terminal sequence of the enzyme establishes that it is encoded by the tcmH gene. The monooxygenase displays an optimal pH of 7.5 and has a Km of 7.47 +/- 0.67 microM and Vmax of 473 +/- 10 nmol.min-1.mg-1. Formally, the Tcm F1 monooxygenase can be classified as an internal monooxygenase that requires only O2 for the enzymatic oxidation. Yet, it apparently does not possess any of the prosthetic groups of known monooxygenases, such as flavin or heme groups, nor does it utilize metal ions. It is inactivated by p-chloromercuribenzoic acid, N-ethylmaleimide, and diethyl pyrocarbonate, suggesting that sulfhydryl groups and histidine residues are essential for the enzyme activity.