The Gram-positive pathogen Listeria monocytogenes encounters acid environments in low-pH foods, during passage through the stomach and within the macrophage phagosome during systemic infection. A novel promoter-trap system termed pGAD-HLY was developed, based on a plasmid containing a promoterless copy of gadB (required for survival at low pH) and hly (whose product facilitates escape from the macrophage phagosome) to identify loci that are induced under different stress conditions in vitro as well as identifying in vivo inducible promoters expressed during intracellular infection. This system facilitated the identification of 11 acid-inducible genes in L. monocytogenes. Transcriptional analysis and acid tolerance response assays confirmed the low-pH induction of these loci, validating this promoter-trap system. Macrophage assays revealed the phagosomal induction of three clones, corresponding to lmo0095, lmo2565 and lmo2371, with two of these clones (lmo0095 and lmo2565) also being induced during murine infection. However, virulence studies did not show any significant difference between strains carrying insertional mutations in these genes and the wild type strain. Although the loci that were identified by this screening procedure do not appear to be central to listerial pathogenesis, it is evident from studies that they contribute to the 'fitness' of this pathogen in adverse acid conditions.