We found that virulent hemolysin-producing (Hly+) Listeria monocytogenes strains inhibit antigen processing and presentation when added to macrophages in vitro. A virulent Hly- bacteria caused little or no inhibition. Live Hly+ bacteria inhibited presentation of both heat-killed L. monocytogenes and ovalbumin. Several observations indicate that hemolysin produced by intracellular bacteria was responsible for the inhibition. First, inhibition was observed even when extracellular bacteria were removed after a brief 10-min bacterial uptake period. Second, inhibition was not prevented by the addition of cholesterol, a substance which inactivates soluble hemolysin. Third, only very high concentrations of soluble hemolysin were inhibitory. Under conditions which inhibit antigen presentation (10(5) per well), macrophages retained normal levels of Ia, maintained normal morphology, and were not permeable when assayed by chromium release. The uptake and catabolism of 35S-labeled live bacteria by macrophages were similar for both Hyl+ and Hly- bacteria. Only a small decrease in uptake and catabolism of surface-iodinated heat-killed L. monocytogenes by macrophages pretreated with inhibitory numbers of live Hly+ bacteria was observed. Additionally, macrophages pretreated with live Hly+ bacteria and fixed 1.5 h later were able to effectively present an ovalbumin peptide (amino acids 323 to 339) to the T-cell hybridoma DO11.10. Hemolysin-producing bacteria inhibited the presentation of antigens that need processing better than they did of antigens that do not require a processing event. Thus, we have demonstrated inhibition of an intracellular antigen processing pathway by hemolysin-producing L. monocytogenes, which may contribute to the virulence of this pathogen.