Listeria monocytogenes, a foodborne pathogen, causes listeriosis in humans and animals. Livern is a major target organ for the bacteria, where they invade and multiply within hepatocytes. There is a need for a better understanding of how in vitro assays relate to in vivo virulence. Therefore, the virulence of 3 food isolates (G3990, G3982 and H7550), 2 primate clinical isolates (12375 and 12443) and a human clinical isolate (Scott A) of Listeria was assessed in murine TIB73 hepatocytes. The virulent hemolysin positve strain (Listeria hyl+) and avirulent hemolysin negative strain (Listeria hly-) were used as controls. Three of the isolates (12375,12443 and G3982) exhibited greater virulence, two (G3990 and Scott A) similar virulence and one (H7550) less virulence in hepatocytes than Listeria hyl+. The isolate 12443, which is the most virulent in vivo, has been found to induce stillbirth in experimentally infected rhesus macaques.