The ruminant pathogen Listeria ivanovii differs from Listeria monocytogenes in that it causes strong, bizonal haemolysis and a characteristic shovel-shaped co-operative haemolytic ('CAMP-like') reaction with Rhodococcus equi. We cloned the gene responsible for the differential haemolytic properties of L. ivanovii, smcL. It encodes a sphingomyelinase C (SMase) highly similar (> 50% identity) to the SMases from Staphylococcus aureus (beta-toxin), Bacillus cereus and Leptospira interrogans. smcL was transcribed monocistronically and was expressed independently of PrfA. Low-stringency Southern blots demonstrated that, within the genus Listeria, smcL was present only in L. ivanovii. We constructed an smcL knock-out mutant. Its phenotype on blood agar was identical to that of L. monocytogenes (i.e. weak haemolysis and no shovel-shaped CAMP-like reaction with R. equi ). This mutant was less virulent for mice, and its intracellular proliferation was impaired in the bovine epithelial-like cell line MDBK. The role of SmcL in intracellular survival was investigated using an L. monocytogenes mutant lacking the membrane-damaging determinants hly, plcA and plcB, being thus unable to grow intracellularly. Complementation of this mutant with smcL on a plasmid was sufficient to promote bacterial intracellular proliferation in MDBK cells. Transmission electron microscopy showed that SmcL mediates the disruption of the phagocytic vacuole and the release of bacteria into the cytosol. Therefore, L. ivanovii possesses a third phospholipase with membrane-damaging activity that, together with PlcA and PlcB, may act in concert with the pore-forming toxin Hly to mediate efficient escape from the vacuolar compartment. The 5' end of smcL is contiguous with the internalin locus i-inlFE, which is also specific to L. ivanovii and is required for full virulence in mice. Thus, smcL forms part of a novel virulence gene cluster in Listeria that is species specific.