The oncolytic effects of herpes simplex virus-1 (HSV-1) are limited, possibly because of premature death of infected cells by apoptosis, which limits the amount of progeny virus that is produced. It has been proposed that inhibition of apoptosis in infected tumor cells would allow increased viral persistence, replication and therapeutic effect. To test this hypothesis, we infected monocyte chemoattractant factor-7 (MCF-7) and MDA-MB-231 breast cancer cells with HSV-1 strain 17(+) and 17Δγ34.5 in the presence or absence of N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVADfmk), a pan-caspase inhibitor. At low doses of HSV-1 strain 17(+) and 17Δγ34.5, the growth of MCF-7 cells was reduced to 37% or 42%, respectively, of uninfected cells. However, when cells were infected in the presence of zVADfmk, cell growth was further reduced to 24 and 33%. Similar results were seen in MDA-MB-231 cells. Cells treated with zVADfmk contained roughly 10 times more infectious viral particles than cells infected without zVADfmk, as shown by both plaque-forming and quantitative polymerase chain reaction assays. To model the situation within an infected tumor, supernatant fluids were collected from infected and non-infected cell cultures and then passed to non-infected cells. In the presence of zVADfmk, the cell growth inhibitory effect became stronger with repeated passages and was attributed to viral replication, because it could be prevented by anti-HSV antibody. These results suggest that caspases represent a novel target for drugs that increase the therapeutic efficacy of oncolytic herpes viruses against breast cancer.