Cerulenin is a potent inhibitor of fatty acid synthase (FAS) in a variety of prokaryotic and eukaryotic cells. Using a standardized mycobacterial susceptibility test, we have observed that cerulenin inhibits the growth of several species of mycobacteria, including tuberculous species such as Mycobacterium tuberculosis (H37Rv and clinical isolates) and Mycobacterium bovis BCG (hereafter called BCG), as well as several non-tuberculous species: Mycobacterium smegmatis, the Mycobacterium avium-intracellulare complex (MAC), Mycobacterium kansasii and others. All species and strains tested, including multi-drug resistant isolates of M. tuberculosis, were susceptible to cerulenin with MICs ranging from 1.5 to 12.5 mg/L. Two-dimensional thin-layer chromatography revealed different inhibition patterns of lipid synthesis between tuberculous and non-tuberculous mycobacteria. Cerulenin treatment resulted in a relative increase in phospholipids and mycolic acids in MAC and M. smegmatis, whereas in cerulenin-treated BCG, phospholipids and mycolic acids diminished relative to controls. In addition, long-chain extractable lipids (intermediate in polarity), triglycerides and glycopeptidolipids decreased with cerulenin treatment in all three species of mycobacteria tested. Qualitative changes in several of these lipid classes indicate inhibition in the synthesis of intermediate and long-chain fatty acids. Our results suggest that cerulenin's primary effect may be inhibition of intermediate and long-chain lipid synthesis, with little effect on the synthesis of other lipid classes. In addition, the BCG-specific reduction in phospholipids and mycolic acids suggests the presence of a unique cerulenin-sensitive FAS system in tuberculous mycobacteria. Since pathogenic mycobacteria produce novel long-chain fatty acids, inhibition of fatty acid synthesis offers a potential target for the development of antimycobacterial drugs.