Listeria monocytogenes insertion mutants defective in hemolysin production were generated using the conjugative transposons Tn916 and Tn1545. All of the nonhemolytic mutants (hly-) lacked a secreted 58-kD polypeptide, presumedly hemolysin, and were avirulent in a mouse model. An intracellular multiplication assay was established in monolayers of mouse bone marrow-derived macrophages, the J774 macrophage-like cell line, the CL.7 embryonic mouse fibroblast cell line, and the Henle 407 human epithelial cell line. The hly+ strain grew intracellularly in all of the tissue culture cells with a doubling time of approximately 60 min. In contrast, the hly- mutants failed to grow in the murine-derived tissue culture cells, but retained the ability to grow in the human tissue culture cells examined. Hemolytic-positive revertants were selected after passage of the hly- mutants through monolayers of J774 cells. In each case, the hemolytic revertants possessed the 58-kD polypeptide, were capable of intracellular growth in tissue culture monolayers and were virulent for mice.