NADPH-Dependent enoyl-CoA reductase [EC 220.127.116.11] was purified to homogeneity, for the first time, from the crude extract of Mycobacterium smegmatis. The molecular weight of this enzyme was estimated to be around 32,000 using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme reduced 2-trans-hexadecenoyl-CoA (Km value, 100 microM) and -eicosenoyl-CoA (Km value, 83 microM) almost equally well in the presence of NADPH as a sole electron donor. The Km value for NADPH was 34.5 microM. When NADP3H was incubated with 2-eicosenoyl-CoA and the purified enzyme, the sole tritiated product was arachidate. This enzyme was almost inert to enoyl-CoAs with chains less than 12 carbon atoms long. The purified enzyme still retained FMN, which was detectable by acid ammonium sulfate and was essential for full activity of the enzyme. The enzyme was sensitive to SH-reagents such as N-ethylmaleimide and monoiodoacetamide but was not sensitive to isonicotinamide hydrazide. Anti-NADPH-dependent-enoyl-CoA-reductase rabbit serum was found to inhibit the activities of both the reductase and the malonyl-CoA dependent fatty acid elongation system, supporting the involvement of the reductase in this elongation system.