The requirements for optimal activity of the hepatitis A virus (HAV) internal ribosome entry segment (IRES) differ substantially from those of other picornavirus IRESes. One such difference is that, to date, the HAV IRES is the only one whose efficiency is severely inhibited in the presence of the picornaviral 2A proteinase. Here we describe experiments designed to dissect the mechanism of proteinase-mediated inhibition of HAV translation. Using dicistronic mRNAs translated in vitro, we show that the HAV IRES is inhibited by the foot-and-mouth disease virus Lb proteinase, as well as by the human rhinovirus 2A proteinase. Furthermore, using mutant Lb proteinase, we demonstrate that proteolytic activity is required for inhibition of HAV IRES activity. Translation inhibition correlated closely with the extent of cleavage of the one identified common cellular target for the 2A and Lb proteinases, eukaryotic initiation factor (eIF) 4G, a component of the eIF4F cap-binding protein complex. Total rescue of HAV IRES activity was possible if purified eIF4F was added to translation extracts. In contrast, if the added eIF4F contained cleaved eIF4G, no rescue of HAV IRES activity was evidenced. Thus the HAV IRES requires intact eIF4G for activity. This is unique among the picornavirus IRESes studied to date and may help explain why HAV does not inhibit host cell translation during viral infection.