Listeria monocytogenes is a foodborne pathogen able to cause serious disease in humans and animals. Not all isolates are equally pathogenic, however, and several isolates have been characterized as naturally virulence attenuated. We sought to identify the genetic basis of natural virulence attenuation using cell culture assays and molecular techniques. By comparing the phenotypes of naturally virulence-attenuated isolates to those of defined virulence gene mutants in plaque, cytotoxicity, and hemolysis assays and by characterizing selected virulence genes and their expression using DNA sequencing and TaqMan reverse transcriptase PCR, we classified virulence-attenuated isolates into four categories. Both group A and group B isolates were noncytotoxic and nonhemolytic; however, group A isolates underexpressed listeriolysin O (LLO, encoded by hlyA), while group B isolates produced LLO proteins that were inactive. The single isolate in group C was fully cytotoxic, had higher than wild-type hemolytic activity, and was, therefore, likely virulence attenuated due to overexpression of LLO. Group D isolates were characterized by normal cytotoxicity, hemolytic activity, and hlyA expression but had reduced intracellular growth. The genetic mechanisms causing virulence-attenuated phenotypes among the group D isolates could not be determined definitively but may involve defects in the expression of actA or the function of the ActA protein. Our results show (i) that the combination of cell culture assays and molecular techniques used in this study allows for identification and characterization of naturally virulence-attenuated isolates and (ii) that multiple distinct genetic mechanisms are responsible for natural virulence attenuation in L. monocytogenes.