Nanaomycin D reductase, catalyzing the conversion of nanaomycin D to nanaomycin A, which is the first step in the biosynthetic sequence (D leads to A leads to E leads to B) in Streptomyces rosa var. notoensis, was purified from the crude extract of the strain by ammonium sulfate fractionation and column chromatography on DEAE-cellulose, Sephadex G-100 and hydroxyapatite to give an electrophoretically homogeneous preparation. The enzyme was found to be a flavoprotein which contains FAD as a prosthetic group and has a molecular weight of 68,000 daltons. It catalyzed the reductive transformation of nanaomycin D to nanaomycin A in the presence of NADH under anaerobic conditions. The Km values were 250 microM for nanaomycin D and 62 microM for NADH. The enzyme was inhibited by 1 mM Cu2+ ion and by NADH at concentrations over 50 microM. The optimal pH was 5.0 and the optimal temperature was 37 degrees C. Several benzoisochromane-quinone antibiotics other than nanaomycin D, kalafungin (enantiomer of nanaomycin D), griseucin A and frenolicin B were converted to the corresponding reduced products by the enzyme. However, granaticin and 4 alpha, 10 alpha-epoxynanaomycin D were not converted.