Intracellular parasites can be classified into those that reside within a host vacuole and those which grow directly in the host cytoplasm. Members of the latter group, which includes Rickettsia, Shigellae, Trypanosoma cruzi, and Listeria monocytogenes, possess haemolytic activity associated with the ability to enter the host cytoplasm. Therefore mutants of L. monocytogenes lacking a pore-forming haemolysin, listeriolysin O, do not escape from the endosomal compartment and consequently fail to become established in the cytoplasm. To examine the role of listeriolysin O, we cloned the structural gene for the L. monocytogenes haemolysin, hlyA, into an asporogenic mutant of Bacillus subtilis under the control of an IPTG-inducible promoter. After being internalized by the macrophage-like cell line J774, haemolytic B. subtilis disrupted the phagosomal membrane and grew rapidly within the macrophage cytoplasm. These results show that a single gene product is sufficient to convert a common soil bacterium into a parasite that can grow in the cytoplasm of a mammalian cell.