The NADH reductase component of the steroid 9alpha-hydroxylase fromMycobacterium fortuitum was purified to homogeneity. Recovery of the enzyme from the 50 approximately 60% ammonium sulfate saturated fraction was 49%, with a purification factor of 100-fold. The NADH reductase has a relative molecular mass of 60 KDa as determined by SDS-PAGE. The absorption maxima at 410 and 450 nm indicate the presence of iron-sulfur group and flavin. These prosthetic groups seemed to function as redox groups that transfer electrons from NADH to the following protein. The K(M) value for NADH as substrate was 68 muM. The NH(2)-terminal amino acid sequence of the reductase was determined as Met-Asp-Ala-Ile-Thr-Asn-Val-Pro-Leu-Pro-Ala-Asn-Glu-Pro-Val-His-Asp-Tyr-Ala-Thr. This sequence does not show a homology with the NH(2)-terminal sequences reported for the reductase component of other monooxygenases, suggesting that the NADH reductase component of the steroid 9alpha-hydroxylase system is novel.