In Listeria monocytogenes the alternative sigma factor sigmaB plays important roles in both virulence and stress tolerance. In this study a proteomic approach was used to define components of the sigmaB regulon in L. monocytogenes 10403S (serotype 1/2a). Using two-dimensional gel electrophoresis and the recently developed isobaric tags for relative and absolute quantitation technique, the protein expression profiles of the wild type and an isogenic delta sigB deletion strain were compared. Overall, this study identified 38 proteins whose expression was sigmaB dependent; 17 of these proteins were found to require the presence of sigmaB for full expression, while 21 were expressed at a higher level in the delta sigB mutant background. The data obtained with the two proteomic approaches showed limited overlap (four proteins were identified by both methods), a finding that highlights the complementarity of the two technologies. Overall, the proteomic data reaffirmed a role for sigmaB in the general stress response and highlighted a probable role for sigmaB in metabolism, especially in the utilization of alternative carbon sources. Proteomic and physiological data revealed the involvement of sigmaB in glycerol metabolism. Five newly identified members of the sigmaB regulon were shown to be under direct regulation of sigmaB using reverse transcription-PCR (RT-PCR), while random amplification of cDNA ends-PCR was used to map four sigmaB-dependent promoters upstream from lmo0796, lmo1830, lmo2391, and lmo2695. Using RT-PCR analysis of known and newly identified sigmaB-dependent genes, as well as proteomic analyses, sigmaB was shown to play a major role in the stationary phase of growth in complex media.