Among the 13 known serovars of Listeria monocytogenes, strains exhibiting the serovar 4b are the most prevalently involved in epidemics of human listeriosis. The molecular reasons for the major involvement of serovar 4b strains in all major foodborne outbreaks in contrast to the lower prevalence of this serovar among food isolates remain indefinite. In order to provide further insight in the protein expression of L. monocytogenes 4b strains, the cytoplasmic and extracellular proteomes of L. monocytogenes 4b strains from different origins, i.e. environmental, clinical and asymptomatical carriage, were investigated by two-dimensional gel electrophoresis. Statistical hierarchical clustering analysis on subproteomic profiles clearly discriminated the strains according to their origin. Protein spots differentiating the subproteome patterns were identified using MALDI-TOF MS through their peptide mass fingerprint.