Streptomyces antibioticus NF-18 is a hyperproducing strain of a Streptomyces hormone, virginiae butanolide A (VB-A), that induces virginiamycin production of S. virginiae at nanomolar concentrations. To characterize the biosynthetic pathway of VB-A, we identified and characterized for the first time the 6-dehydro VB-A reductase that is responsible for the final reduction step in the biosynthesis. Assay protocols and stabilization conditions were established. The 6-dehydro VB-A reductase was found to require NADPH, not NADH, as a coenzyme. The K(m) values of the enzyme for NADPH and (+/-)-6-dehydro VB-A were determined to be 50 +/- 2 microM and 100 +/- 5 microM, respectively. Ultracentrifugation experiments revealed that 6-dehydro VB-A reductase was present almost exclusively in the 100,000 x g supernatant fraction, indicating that the enzyme is a cytoplasmic-soluble protein. The M(r) of the native 6-dehydro VB-A reductase was estimated to be 82,000 +/- 3000 by molecular sieve HPLC. The optimal pH was found to be 6.7 +/- 0.2.