Mycothiol is the major low-molecular-weight thiol found in actinomycetes, including Mycobacterium tuberculosis, and has important antioxidant and detoxification functions. Gene disruption studies have shown that mycothiol is essential for the growth of M. tuberculosis. Because of mycothiol's unique characteristics, inhibitors directed against mycothiol biosynthesis have potential as drugs against M. tuberculosis. Four genes have been identified in mycobacteria that are involved in the biosynthesis of mycothiol. Two genes, mshB and mshD, are not essential for growth of M. tuberculosis. Mutants in these genes produce significant amounts of mycothiol or closely related thiol compounds. A targeted gene disruption in the mshC gene is lethal for M. tuberculosis, indicating that MshC is essential for growth. The remaining gene, mshA, encodes for a glycosyltransferase. In the present study, we attempted to produce a directed knock-out of the mshA gene in M. tuberculosis Erdman but found that this was only possible when a second copy of mshA was first incorporated into the chromosome. Bacteria with only a single copy of mshA that grew after mutagenesis produced normal levels of mycothiol. We therefore conclude that the mshA gene, like the mshC gene, is essential for the growth of M. tuberculosis.