The alternative sigma factor σB of Listeria monocytogenes is responsible for regulating the transcription of many of the genes necessary for adaptation to both food-related stresses and to conditions found within the gastrointestinal tract of the host. The present study sought to investigate the influence of growth phase and temperature on the activation of σB within populations of L. monocytogenes EGD-e wild-type, ΔsigB, and ΔrsbV throughout growth at both 4°C and 37°C, using a reporter fusion that couples expression of EGFP to the strongly σB-dependent promoter of lmo2230. A similar σB activation pattern within the population was observed in wt-egfp at both temperatures, with the highest induction of σB occurring in the early exponential phase of growth when the fluorescent population rapidly increased, eventually reaching the maximum in early stationary phase. Interestingly, induction of σB activity was heterogeneous, with only a proportion of the cells in the wt-egfp population being fluorescent above the background autofluorescence level. Moreover, significant RsbV-independent activation of σB was observed during growth at 4°C. This result suggests that an alternative route to σB activation exists in the absence of RsbV, a finding that is not explained by the current model for σB regulation.