ABSTRACT Transcriptional regulation of genes involved in the biosynthesis of cell wall lipids of Mycobacterium tuberculosis is poorly understood. The gene encoding mycocerosic acid synthase ( mas ) and fadD28 , an adjoining acyl coenzyme A synthase gene, involved in the production of a virulence factor, dimycocerosyl phthiocerol, were cloned from Mycobacterium bovis BCG, and their promoters were analyzed. The putative promoters were fused to the xylE reporter gene, and its expression was measured in Escherichia coli , Mycobacterium smegmatis , and M. bovis BCG. In E. coli , the fadD28 promoter was not functional but the mas promoter was functional. Both fadD28 and mas promoters were functional in M. smegmatis , at approximately two- and sixfold-higher levels, respectively, than the BCG hsp60 promoter. In M. bovis BCG, the fadD28 and mas promoters were functional at three- and fivefold-higher levels, respectively, than the hsp60 promoter. Primer extension analyses identified transcriptional start points 60 and 182 bp upstream of the translational start codons of fadD28 and mas , respectively. Both promoters contain sequences similar to the canonical −10 and −35 hexamers recognized by the σ 70 subunit of RNA polymerase. Deletions of the upstream regions of both genes indicated that 324 bp of the fadD28 and 228 bp of the mas were essential for promoter activity. Further analysis of the mas promoter showed that a 213-bp region 581 bp upstream of the mas promoter acted as a putative transcriptional enhancer, promoting high-level expression of the mas gene when present in either direction. This represents the identification of a rare example of an enhancer-like element in mycobacteria.