Cytotoxic cells provide a crucial defense against DNA and RNA viral infections. Here we describe an in vitro model to study the fate of vesicular stomatitis virus (VSV) RNA in cells undergoing apoptosis. Using the [3H]uridine release assay, we show that human LAK cells induce the degradation of RNA in infected U937 cells in addition to inhibiting the production of infectious virions. LAK cell-mediated RNA degradation was blocked by the serine protease inhibitor, 3,4-dichloroisocoumarin. Purified human granzyme B but not inactivated granzyme B, granzyme A, or perforin rapidly induced degradation of RNA in VSV-infected U937 cells in a dose- and time-dependent manner without lysing the cells and suppressed viral production. Northern analysis of RNA extracted from infected cells with a VSV full-length cDNA probe confirmed that levels of viral transcripts were reduced by treatment with granzyme B. Nevertheless, the amount of host beta-actin mRNA was also reduced in infected cells, suggesting that treatment with granzyme B induced apoptosis. Consistent with this notion, infected cells exposed to granzyme B rapidly developed DNA strand breakage. Taken together, the data suggest that granzyme B in the absence of perforin reduced VSV production by activating a mechanism that degraded viral transcripts in infected U937 cells.