Certain picornaviruses encode proteinases which cleave the translation initiation factor eIF4G, a member of the eIF4F complex which recruits mRNA to the 40S ribosomal subunit during initiation of protein synthesis in eukaryotes. We have compared the efficiency of eIF4G cleavage in rabbit reticulocyte lysates during translation of mRNAs encoding the foot-and-mouth disease virus leader proteinase (Lpro) or the human rhinovirus 2Apro. Under standard translation conditions, Lpro cleaved 50% of eIF4G within 4 min after initiation of protein synthesis, whereas 2Apro required 15 min. At these times, the molar ratios of proteinase to eIF4G were 1:130 for Lpro and 1:12 for 2Apro, indicating a much more efficient in vitro cleavage than previously observed. The molar ratios are similar to those observed during viral infection in vivo.