The role of the alternative sigma(54) factor, encoded by the rpoN gene, was investigated in Listeria monocytogenes by comparing the global gene expression of the wild-type EGDe strain and an rpoN mutant. Gene expression, using whole-genome macroarrays, and protein content, using two-dimensional gel electrophoresis, were analysed. Seventy-seven genes and nine proteins, whose expression was modulated in the rpoN mutant as compared to the wild-type strain, were identified. Most of the modifications were related to carbohydrate metabolism and in particular to pyruvate metabolism. However, under the conditions studied, only the mptACD operon was shown to be directly controlled by sigma(54). Therefore, the remaining modifications seem to be due to indirect effects. In parallel, an in silico analysis suggests that sigma(54) may directly control the expression of four different phosphotransferase system (PTS) operons, including mptACD. PTS activity is known to have a direct effect on the pyruvate pool and on catabolite regulation. These results suggest that sigma(54) is mainly involved in the control of carbohydrate metabolism in L. monocytogenes via direct regulation of PTS activity, alteration of the pyruvate pool and modulation of carbon catabolite regulation.