Mycobacterium bovis BCG produces a variety of methyl-branched fatty acids. They include C28 to C32 mycocerosic acids esterified to phthiocerol and phenolphthiocerol and the shorter (C22 to C26) mycocerosic acids esterified to phthiocerol. A mycocerosic acid synthase gene-disrupted mutant was still able to produce the shorter mycocerosic acids. The enzyme short chain mycocerosic acid synthase (SMAS), that catalyzes the synthesis of such acids, was purified using anion exchange and red-agarose chromatography. Gel filtration showed the native enzyme to be a 537-kDa protein. Since SDS-polyacrylamide gel electrophoresis of the purified enzyme showed a 280-, 170-, and 100-kDa protein and they cross-reacted with antibodies prepared against the 280- or 100-kDa protein, this enzyme is composed of the three subunits or two 280-kDa monomers with an unusual susceptibility to a proteolytic nick. SMAS utilizes methylmalonyl-CoA with C12 to C20 acyl-CoA as primers and with either NADH or NADPH as the reductant to synthesize the short mycocerosic acids. The Km values for NADH and NADPH were 93 and 90 microM, respectively. Antibodies raised against either the 280- or 100-kDa protein inhibited the incorporation of methylmalonyl-CoA into fatty acids by SMAS. The enzyme is not immunologically closely related to mycocerosic acid synthase or fatty acid synthase.