Lipids that are found only in the cell envelope of pathogenic mycobacteria, such as those containing multiple methyl-branched fatty acids, have long been thought to play a role in pathogenesis. Among these complex lipids, sulfolipids have been the most extensively studied over the last 50 years. The numerous biological effects exhibited by purified sulfolipids on phagocytic cells led to the idea that these molecules are probably important virulence factors facilitating the intracellular survival of Mycobacterium tuberculosis. However, definitive evidence to support this concept has been lacking. The recent construction of an isogenic sulfolipid-deficient mutant of M. tuberculosis H37Rv (Sirakova et al., J. Biol. Chem. 276:16833-16839, 2001) has for the first time provided the opportunity to directly assess the contribution of these complex lipids to pathogenesis. In the present study, we show that against all expectations, sulfolipid deficiency does not significantly affect the replication, persistence, and pathogenicity of M. tuberculosis H37Rv in mice and guinea pigs or in cultured macrophages.Tuberculosis remains a major health problem worldwide (26). Mycobacterium tuberculosis, the causative agent of this disease in humans, has the capacity to infect, replicate in, and persist inside host macrophages. The virulence factors enabling M. tuberculosis to escape the bactericidal mechanisms of these cells, notably by blocking phagosome-lysosome fusion, are essentially unknown (6). A study conducted some 55 years ago by Middlebrook and collaborators (21) recognized that the curious ability of the tubercle bacillus to grow in culture as serpentine cords was relatively restricted to virulent strains. Dubos and Middlebrook (7) also observed that the virulent cord-forming strains invariably absorbed the cationic dye neutral red, while most attenuated strains of M. tuberculosis and saprophytic mycobacteria did not.In a search for the component of virulent mycobacteria that induced uptake of the dye, Middlebrook and coworkers (20) isolated sulfolipids. This family of molecules is composed of sulfated trehalose esters acylated with three to four acyl groups consisting of one short saturated fatty acid (palmitic acid or stearic acid) and different combinations of long-chain multiple methyl-branched fatty acids (the phthioceranic and hydroxyphthioceranic acids) (for a review, see references 9 and 11). Consistent with their suspected role in virulence, sulfolipids are found only in the human pathogen M. tuberculosis (9). Interestingly, sulfolipids are present in the virulent laboratory strain M. tuberculosis H37Rv but absent from the avirulent strain M. tuberculosis H37Ra (20).