Listeria monocytogenes strains previously generated by transposon mutagenesis were examined with respect to virulence and induction of protective immunity in BALB/c mice. The phenotypic defects observed in these mutant L. monocytogenes strains included decreased hemolysin (listeriolysin O [LLO]) production, phospholipase C activity, intracellular growth, and/or cell-to-cell spread in vitro. While 50% lethal dose determinations performed with these mutant strains indicated reduced virulence for BALB/c mice, sublethal infection with the majority of these mutant strains provided protection against a subsequent challenge with the fully virulent L. monocytogenes parent strain. In addition, in vitro infection of the J774 cell line with most of these mutant strains converted these phagocytic cells to targets of L. monocytogenes-immune cytotoxic cells. The exceptions to these findings were two LLO-negative, avirulent mutant strains which were unable to immunize mice against a secondary challenge with virulent L. monocytogenes. One of these mutants contained a transposon insertion within the structural gene for LLO, and the other contained a transposon insertion in the structural gene for the transcriptional activator of the LLO gene. These two LLO-negative mutant strains also were unable to escape phagolysosomes in infected J774 cells and could not transform these phagocytic cells into targets of L. monocytogenes-immune cytotoxic cells. These findings confirm the importance of LLO in the induction of antilisterial immunity and suggest that a cytoplasmic localization of these pathogenic bacteria is required for the development of protective immunity.