Listeria monocytogenes σBand PrfA are pleiotropic regulators of stress response and virulence gene expression. Quantitative RT-PCR (qRT-PCR) was used to measure transcript levels ofσB- and PrfA-dependent genes in exponential-phaseL. monocytogeneswild-type and ΔsigBstrains as well as in bacteria exposed to environmental stresses (0.3 M NaCl or growth to stationary phase) or present in the vacuole or cytosol of human intestinal epithelial cells. Stationary-phase or NaCl-exposedL. monocytogenesshowedσB-dependent increases inopuCA(10- and 17-fold higher, respectively) andgadAtranscript levels (77- and 14-fold higher, respectively) as compared to non-stressed, exponential-phase bacteria. While PrfA activity, as reflected byplcAtranscript levels, was up to 95-fold higher in intracellularL. monocytogenesas compared to non-stressed bacteria,σBactivity was only slightly higher in intracellular than in non-stressed bacteria. IncreasedplcAtranscript levels, which were similar in both host cell vacuole and cytosol, were associated with increases in bothprfAexpression and PrfA activity. qRT-PCR assays were designed to measure expression ofprfAfrom each of its three promoter regions. Under all conditions, readthrough transcription from the upstreamplcApromoter was very low. The relative contribution to totalprfAtranscription from theσA-dependent P1prfApromoter ranged from ∼17 % to 30 %, while the contribution of the P2prfAregion, which appears to be transcribed by bothσAandσB, ranged from ∼70 % to 82 % of totalprfAtranscript levels. In summary (i)σBis primarily activated during environmental stress and does not contribute to PrfA activation in intracellularL. monocytogenesand (ii) the partiallyσB-dependent P2prfApromoter region contributes the majority ofprfAtranscripts in both intra- and extracellular bacteria.