Human enteroviruses and rhinoviruses rapidly and selectively abolish translation from cellular mRNA upon infection of susceptible cells. Expression of the poliovirus 2A protease (PV 2A pro ) is sufficient to cause host translation shutoff through cleavage of eIF4G (formerly p220, eIF4γ) either directly or indirectly through activation of a cellular factor. Evidence exists for both direct and indirect cleavage mechanisms; however, factors presumed to participate in an indirect mechanism have not yet been purified or defined. Here we show that the dominant eIF4G cleavage activity in lysates from infected HeLa cells was separable from PV 2A pro by size exclusion chromatography. 2A pro separated into two peak fractions which contained activity which cleaved a peptide substrate derived from the poliovirus polyprotein. These peak 2A pro fractions did not cleave eIF4G or an eIF4G-derived peptide, as expected, due to the poor efficiency of direct cleavage reactions. Conversely, fractions which contained peak eIF4G cleavage activity and only trace amounts of 2A pro efficiently cleaved a peptide substrate derived from the previously mapped eIF4G cleavage site and also cleaved a peptide derived from the poliovirus 1D2A region. The dominant eIF4G cleavage activity was highly purified through four chromatography steps and found to be devoid of all traces of 2A pro or its precursors. Quantitation of 2A pro from lysates of infected cells showed that during infections in HeLa cells, 2A pro does not reach molar excess over eIF4G, as previously shown to be required for direct eIF4G cleavagein vitro.Further, infection of HeLa cells in the presence of 2 mM guanidine–HCl, a potent inhibitor of viral RNA replication, suppressed accumulation of 2A pro and its precursor 2ABC below detectable levels but was unable to delay the onset of eIF4G proteolysisin vivo.The eIF4G cleavage activity was still easily detectable inin vitroassays using fractions from guanidine-treated cells. Thus, the data suggest that poliovirus utilizes two catalytic activities to ensure rapid cleavage of eIF4Gin vivo.Although it was not directly measurable here, 2A pro likely does cleave a portion of eIF4G in cells. However, the data suggest that a cellular factor which can be activated by small quantities of 2A pro constitutes the bulk of the eIF4G-specific cleavage activity in infected cells and is responsible for the rapid and efficient eIF4G cleavage activity observedin vivo.