The ability of Salmonella typhimurium to survive and replicate within murine macrophages is dependent on a low phagosomal pH. This requirement for an acidic vacuole suggests that low pH is an important environmental stimulus for the transcription of genes necessary for intracellular survival. To study the behaviour of acid-inducible genes in response to the phagosomal environment, we have applied a novel enrichment strategy, termed differential fluorescence induction (DFI), to screen an S. typhimurium library for promoters that are upregulated at pH 4.5. DFI utilizes a fluorescence-enhanced green fluorescent protein (GFP) and a fluorescence-activated cell sorter (FACS) to perform genetic selection. In the presence of an inducing stimulus, such as low pH, a FACS is used to sort highly fluorescent bacterial clones bearing random promoters fused to the mutant GFP protein (GFPmut). This population is then amplified at neutral pH and the least fluorescent population is sorted. Sequential sorts for fluorescent and non-fluorescent bacteria in the presence or absence of inducing conditions rapidly enriches for promoter fusions that are regulated by the inducing stimulus. We have identified eight acid-inducible promoters and quantified their expression in response to pH 4.5 and to the phagosome milieu. These acid-inducible promoters exhibited extensive homology to promoter regions of genes encoding for cell-surface-maintenance enzymes, stress proteins, and generalized efflux pumps. Only a subset of these promoters was induced in macrophages with kinetics and levels of expression that do not necessarily correlate with in vitro pH-shock induction. This suggests that while low pH is a relevant inducer of intracellular gene expression, additional stimuli in the macrophage can modulate the expression of acid-inducible genes.