Previously, the purified recombinant 2A proteases (2A pro ) of coxsackievirus B4 (CVB4) and human rhinovirus type 2 (HRV2) were shown to cleave synthetic peptides derived from human or rabbit eIF4G as well as eIF4G protein purified from rabbit reticulocytes. These results were in contrast to previous evidence which supported the view that eIF4G cleavage activity in poliovirus-infected HeLa cells required a cellular factor(s) activated by poliovirus (PV) 2A pro . In the present study, recombinant PV 2A pro was shown to cleave either rabbit or human eIF4G or their derived peptides in direct cleavage reactions, but cleaved the 4G-derived peptides with 100-fold lower efficiency than with a peptide derived from the poliovirus polyprotein. In these experiments, up to 25-fold molar excess of 2A pro over eIF4G protein was required to cause greater than 50% cleavage. CVB4 2A pro was also tested in peptide cleavage assays under the same conditions as PV 2A pro and was found to cleave all eIF4G substrates with efficiencies similar to PV 2A pro . Finally, cleavage reactions utilizing recombinant eIF4G containing a G486E substitution at the cleavage site for CVB4 and HRV2 proteases resulted in drastically reduced cleavage by PV 2A pro , similar to the reduction previously seen with HRV2 and CVB4 2A pro , confirming that all three viral 2A proteases recognize the same cleavage site on eIF4G. These data show that PV 2A pro can directly cleave eIF4Gin vitrowith efficiencies similar to those of CVB 2A pro , but cleavage efficiency of eIF4G is approximately 1000-fold lower than cleavage of a peptide derived from the authentic 2A cleavage site on the poliovirus polyprotein.