Under stress conditions, the facultative intracellular pathogen Listeria monocytogenes produces a ClpC ATPase, which is a general stress protein encoded by clpC and belonging to the HSP-100/Clp family. A ClpC-deficient mutant was obtained by gene disruption in strain LO28, which became highly susceptible to stress conditions in vitro. Intracellular growth of this mutant was restricted within macrophages, one of the major target cells of L. monocytogenes, during the infectious process. A quantitative electron microscope study showed that, contrary to wild-type bacteria that rapidly gain access to the cytoplasm of macrophages, mutant bacteria remained confined to membrane-bound phagosomes. Only a few mutant bacteria disrupted the phagosome membrane after 4h of incubation, then polymerized actin filaments and multiplied within the cytoplasm. The ClpC ATPase, therefore, promotes early bacterial escape from the phagosome of macrophages, thus enhancing intracellular survival. The ClpC ATPase was produced in vivo during experimental infection by wild-type bacteria. The virulence of the ClpC-deficient mutant was severely attenuated in mice, with a three-log decrease in its 50% lethal dose compared with wild-type bacteria. Bacterial growth of mutant bacteria was strongly restricted in organs, presumably because of an impairment of intracellular survival in host tissues. Our results provide evidence that a general stress protein is required for the virulence of L. monocytogenes, which behaves as a virulence factor promoting intracellular survival of this pathogen.