The NADH reductase component of the steroid 9.alpha.-hydroxylase from Mycobacterium fortuitum was purified to homogeneity. Recovery of the enzyme from the 50-60% ammonium sulfate saturated fraction was 49%, with a purification factor of 100-fold. The NADH reductase has a relative molecular 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 $68{mu}M$. 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 9.alpha.-hydroxylase system is novel.