The eIF2alpha kinases have been involved in the inhibition of vesicular stomatatis virus replication but the contribution of each kinase to this process has not been fully investigated. Using mouse embryonic fibroblasts (MEFs) from knock-out mice we show that PKR and HRI have no effects on VSV replication as opposed to PERK and GCN2, which exhibit strong inhibitory effects. When MEFs containing the serine 51 to alanine mutation of eIF2alpha were used, we found that VSV replication is independent of eIF2alpha phosphorylation. Nevertheless, the kinase domain of the eIF2alpha kinases is both necessary and sufficient to inhibit VSV replication in cultured cells. Induction of PI3K-Akt/PKB pathway by eIF2alpha kinase activation plays no role in the inhibition of VSV replication. Our data provide strong evidence that VSV replication is not affected by eIF2alpha phosphorylation or downstream effector pathways such as the PI3K-Akt/PKB pathway. Thus, the anti-viral properties of eIF2alpha kinases are not always related to their inhibitory effects on host protein synthesis as previously thought and are possibly mediated by phosphorylation of proteins other than eIF2alpha.