Mycocerosyl lipids are found uniquely in the cell walls of pathogenic mycobacteria. Mycocerosic acid synthase (MAS) is a multifunctional protein which catalyzes elongation of n-fatty acyl-CoA with methylamalonyl-CoA as the elongating agent (Rainwater, D. L., and Kolattukudy, P. E. (1985) J. Biol. Chem. 260, 616-623). To understand how the various domains that catalyze the reactions involved in chain elongation are organized, mas gene from Mycobacterium tuberculosis bovis BCG was cloned. A lambda gt11 library of AluI partially digested genomic DNA from the organism was screened with an oligonucleotide probe designed from the N-terminal amino acid sequence of purified MAS. Using terminal segments of inserts from positive clones as the probe, the library was rescreened and the process was repeated. Sequencing of four overlapping clones revealed a contiguous sequence of 9699 base pair(s) (bp) of mycobacterial genome containing a 6330-bp open reading frame that could code for a protein of 2100 amino acids with a molecular mass of 225,437 daltons. The authenticity of the open reading frame as that of MAS was verified by correspondence of the amino acid sequences deduced from the gene with the directly determined amino acid sequences of the N terminus and three different internal peptide fragments. By comparing the MAS amino acid sequence with the sequences in the active site regions of known fatty acid synthases and polyketide synthases the functional domains in MAS were identified. This analysis showed that the domains were organized in the following order: beta-ketoacyl synthase, acyl transferase, dehydratase-enoyl reductase, beta-ketoreductase, acyl carrier protein; no thioesterase-like domain could be found. These results establish MAS as the first case of an elongating multifunctional enzyme composed of two identical subunits that resemble the vertebrate fatty acid synthase in size, subunit structure, and linear organization of functional domains. Southern and Western blot analyses showed absence of mas gene and encoded proteins in Mycobacterium smegmatis and Escherichia coli. This result is consistent with the report that mycocerosic acid is present only in pathogenic mycobacteria.