As part of the Sec translocase, the accessory ATPase SecA2 is present in some pathogenic Gram-positive bacteria. In Listeria monocytogenes, deletion of secA2 results in filamentous cells that form rough colonies and have lower virulence. However, only a few proteins have been identified that are secreted by this pathway. This investigation aims to provide the first exoproteomic analysis of the SecA2-dependent secretion in L. monocyto genes EGD-e. By using media and temperatures relevant to bacterial physiology, we demonstrated that the rough colony and elongated bacterial cell morphotypes are highly dependent on growth conditions. Subsequently, comparative exoproteomic analyses of the Delta secA2 versus wt strains were performed in chemically defined medium at 20 degrees C and 37 degrees C. Analyzing the proteomic data following the secretomics-based method, part of the proteins appeared routed towards the Sec pathway and exhibited an N-terminal signal peptide. For another significant part, they were primarily cytoplasmic proteins, thus lacking signal peptide and with no predictable secretion pathway. In total, 13 proteins were newly identified as secreted via SecA2, which were essentially associated with cell-wall metabolism, adhesion and/or biofilm formation. From this comparative exoproteomic analysis, new insights into the L. monocytogenes physiology are discussed in relation to its saprophytic and pathogenic lifestyle