2-Enoyl-CoA reductase was purified 150-fold from the crude extract of Mycobacterium smegmatis. The purified reductase required NADH, but not NADPH, as a reductant and catalyzed the reduction of C4 to C16 enoyl-CoAs, though the activities toward shorter chain substrates (c4 and C6) were very low. Thiolase was also partially purified from the same source. These two enzymes were used, together with 3-hydroxyacyl-CoA dehydrogenase and the two forms of enoyl-CoA hydratase (hydratases I and II) previously purified from the same source, to reconstitute fatty acid elongation activity. The products formed from [1-14C]acetyl-CoA and decanoyl-CoA in this reconstituted system were analyzed by thin-layer chromatography and radio-gas-liquid chromatography. The system containing hydratase II produced laurate and 3-hydroxylaurate (in the form of their CoA esters) and the ratio of laurate to 3-hydroxylaurate increased as the incubation time was increased. The system containing hydratase I produced only 3-hydroxylaurate. 3-Hydroxylaurate was also the only product when enoyl-CoA reductase was omitted from the system containing hydratase II. It is concluded that hydratase II, but not hydratase I, is functional in fatty acid elongation by M. smegmatis and that enoyl-CoA reductase is also essential for the reaction. CoA and NAD+ inhibited the reconstituted elongation activity in competition with acetyl-CoA and NADH, respectively.