The initiation of translation on eukaryotic mRNA is governed by the concerted action of polypeptides of the eIF-4F complex. One of these polypeptides, eIF-4G, is proteolytically inactivated upon infection with several members of the Picornaviridae family. This cleavage occurs by the action of virus-encoded proteinases: 2Apro (entero- and rhinovirus) or Lpro (aphthovirus). An indirect mode of eIF-4G cleavage through the activation of a second cellular proteinase has been proposed in the case of poliovirus. Although cleavage of eIF4G by rhino- and coxsackievirus 2Apro has been achieved directly in vitro, a similar activity has not been documented to date for poliovirus 2Apro. We report here that a recombinant form of poliovirus 2Apro fused to maltose binding protein (MBP) directly cleaves human eIF-4G from a highly purified eIF-4F complex. Efficient cleavage of eIF-4G requires magnesium ions. The presence of other initiation factors such as eIF-3, eIF-4A or eIF-4B mimics in part the stimulatory effect of magnesium ions and probably stabilizes the cleavage products of eIF-4G generated by 2Apro. These results suggest that efficient cleavage of eIF4G by MBP-2Apro requires a proper conformation of this factor. Finally, MBP-2Apro protein cleaves an eIF-4G-derived synthetic peptide at the same site as rhino- and coxsackievirus 2Apro (R485-G486).