Lipooligosaccharides (LOS) are highly antigenic glycolipids produced by a number of Mycobacterium species, which include "M. canettii," a member of the M. tuberculosis complex, and the opportunistic pathogens M. marinum and M. kansasii. The various LOS share a core composed of trehalose esterified by at least 1 mole of polymethyl-branched fatty acid (PMB-FA) and differ from one another by their oligosaccharide extensions. In this study, we identified a cluster of genes, MSMEG_4727 through MSMEG_4741, likely involved in the synthesis of LOS in M. smegmatis. Disruption of MSMEG_4727 (the ortholog of pks5 of M. tuberculosis), which encodes a putative polyketide synthase, resulted in the concomitant abrogation of the production of both PMB-FA and LOS in the mutant strain. Complementation of the mutant with the wild-type gene fully restored the phenotype. We also showed that, in contrast to the case for "M. canettii" and M. marinum, LOS are located in deeper compartments of the cell envelope of M. smegmatis. The availability of two mycobacterial strains differing only in LOS production should help in defining the biological role(s) of this important glycolipid.