A better understanding of mycobacterial gene regulation under certain stress conditions (e.g., low pH) may provide insight into mechanisms of adaptation during infection. To identify mycobacterial promoters induced at low pH, we adapted the recombinase-based in vivo expression technology (RIVET) promoter trap system for use with mycobacteria. Our results show that the TnpR recombinase of transposon gammadelta is active in Mycobacterium smegmatis and Mycobacterium tuberculosis. We developed a method to perform sequential double selection with mycobacteria by using RIVET, with a kanamycin preselection and a sucrose postselection. A library of M. tuberculosis DNA inserted upstream of tnpR was created, and using the double selection, we identified two promoters which are upregulated at low pH. The promoter regions drive the expression of a gene encoding a putative lipase, lipF (Rv3487c), as well as a PE-PGRS gene, Rv0834c, in a pH-dependent manner in both M. smegmatis and M. tuberculosis. The acid inducibility of lipF and Rv0834c was independent of the stress response sigma factor, SigF, as acid induction of the two genes in an M. tuberculosis sigF mutant strain was similar to that in the wild-type strain. No induction of lipF or Rv0834c was observed during infection of J774 murine macrophages, an observation which is in agreement with previous reports on the failure of phagosomes containing M. tuberculosis to acidify.