Multidrug-resistant tuberculosis is a major global health emergency. Cell wall lipids of Mycobacterium tuberculosis can play crucial roles in the pathogenesis. The enzymes involved in their synthesis can be ideal new drug targets against tuberculosis, because many such lipids are unique to this pathogen. A variety of multiple methyl-branched fatty acids are among such unique lipids. We have identified seven genes highly homologous to the mas gene, which is known to be involved in the production of one class of such multiple methyl-branched fatty acids. One of these mas-like genes, pks2, was disrupted using a phage-mediated delivery of the disruption construct. Gene disruption by homologous recombination was confirmed by polymerase chain reaction analysis of the flanking regions of the introduced disrupted gene and by Southern analysis. Thin-layer and radio gas-chromatographic analyses of lipids derived from [1-14C]propionic acid and gas chromatography/mass spectrometry analysis of the fatty acids and hydroxy fatty acids showed that the pks2 mutant was incapable of producing hepta- and octamethyl phthioceranic acids and hydroxyphthioceranic acids that are the major acyl constituents of sulfolipids. Consequently, pks2 mutant does not produce sulfolipids. Sulfolipid deficiency in pks2 mutant was confirmed by two-dimensional thin-layer chromatographic analysis of lipids derived from [1-14C]propionic acid and 35SO4(-2). With this sulfolipid-deficient mutant, it should be possible to test for the postulated important roles for sulfolipids in the pathogenesis of M. tuberculosis.