Listeriolysin O (LLO) is a pore-forming cytolysin that enables Listeria monocytogenes to escape from a host cell vacuole. The structural gene for the related cytolysin perfringolysin O (pfo) was cloned downstream from the promoter for hly, the gene encoding LLO, both on a plasmid and on the L. monocytogenes chromosome. Both strains secreted active PFO, although regulation was not identical to that of LLO. The chromosomal PFO-expressing strain was characterized for intracellular growth and cell-to-cell spread. It escaped from a host cell vacuole with 64% efficiency compared with the wild type as determined by immunofluorescent staining of bacteria for F-actin, a marker for entry into the cytoplasm. In addition, it replicated intracellularly with a doubling time similar to that of the wild type for 5 h, after which growth was aborted because of a cytotoxic effect on the host cell and influx of extracellular gentamicin. The chromosomal PFO strain was able to plaque in mouse L2 fibroblasts, but it did so at 20% efficiency compared with the wild type and the plaques were significantly smaller. Both strains expressing PFO were completely avirulent in mice. These results indicate that PFO can mediate escape from a host cell vacuole but cannot complement an hly deletion strain for virulence.