By using cell-free systems prepared from uninfected and poliovirus-infected cells, we have been able to demonstrate that crude preparations of initiation factors from infected cells do not stimulate the initiation of translation by polyribosomes containing endogenous host cell mRNA. When tested with polysomes containing endogenous viral mRNA, however, they were able to stimulate initiation of translation nearly as well as uninfected cell initiation factors. The uninfected cell initiation factor preparations were able to stimulate initiation of translation of both cell and viral mRNA. The results indicate an mRNA-specific activity present in crude initiation factor preparations from infected cells. Furthermore, the ability of eIF2 from infected cells to form a ternary complex with GTP and formyl [35S]methionine-tRNAfmet, an mRNA-independent step in initiation, was found not to be deficient. Implications of these data for proposed mechanisms of poliovirus-induced host cell shutoff are discussed.