The survival mechanism of dormant tubercle bacilli is unknown; however, accumulating evidence indicates that Mycobacterium tuberculosis can survive and persist in hypoxic and mildly acidic microenvironments. Such conditions are found in the acidic vacuoles of macrophages, which M. tuberculosis is known to target. We used DECAL (differential expression using customized amplification library) to identify the genes expressed under acidic and hypoxic conditions, following the cultivation of M. tuberculosis H37Rv at an acidic pH and/or under hypoxic or anoxic conditions in vitro. Of 960 clones analysed, 144 genes, consisting of 71 induced and 8 repressed genes, were identified by sequencing and divided into functional categories to characterize their cellular roles. In general, the genes induced under acidic and hypoxic conditions were involved in the biosynthesis of secondary metabolites (e.g. pks4), lipid metabolism, energy production (e.g. pckA) and cell wall biogenesis (e.g. Rv0696 and plcB). The combination of genes identified may explain the energy processing and energy storage of M. tuberculosis during latent infection. These findings not only enhance our understanding of the mechanism of dormancy, but they also may be useful in the design of therapeutic tools and vaccines for latent tuberculosis.