Cytotoxic T lymphocytes (CTL) induce in target cells a rapid, prelytic fragmentation of target cell DNA, accompanied by apoptosis. In contrast, complement and (with a few exceptions) chemical and physical means of inducing cytolysis induce necrosis, without DNA fragmentation. The function of the unusual DNA fragmentation induced by CTL remains to be elucidated. The major recognized function of CTL is in halting virus infections. Earlier, we proposed that CTL might halt virus infections prelytically, by fragmenting viral and cellular nucleic acids, and that in this case, cytolysis per se might be a less important function of CTL. We report here experiments designed to detect prelytic halt of virus replication. We employed in vivo-like conditions: fibroblast targets (difficult to lyse) were infected with herpes simplex virus (HSV), then incubated at low E/T cell ratios overnight. At the highest E/T ratios which produced less than 10% CTL-induced lysis, plaque-forming unit yield was reduced by about 50%. At higher E/T ratios which lysed 1/6 to 1/3 of the infected target cells, 3/4 to 9/10 of the virus production was prevented. The discrepancy between the level of lysis and the reduction in virus yield is evidence for significant CTL-induced prelytic halt of HSV replication. At present, it is unclear whether the antiviral effect observed involves an activity of CTL distinct from their lytic ability, such as their DNA fragmenting ability.