The 2A proteinase (2A(pro)) of human rhinoviruses (HRVs) is a cysteine protease containing a structurally important zinc ion. In the viral polyprotein, the enzyme cleaves between the C terminus of VP1 and its own N terminus. 2A(pro) also processes the two isoforms of the cellular protein, eukaryotic initiation factor 4G (eIF4G). We have shown that mature HRV2 2A(pro), when translated in vitro in rabbit reticulocyte lysates, efficiently cleaves eIF4GI, although the enzyme was not immediately active upon synthesis. Here, we examine the relationship between self-processing and eIF4GI cleavage. The onset of both reactions first occurred at least 10 min after initiation of protein synthesis. Furthermore, when self-processing was prevented by a specific mutation between VP1 and 2A(pro), the VP1-2A(pro) precursor was essentially unable to cleave eIF4GI, implying that self-processing is a prerequisite for eIF4GI cleavage. 2A(pro) synthesized in the presence of a potent zinc chelator is inactive; however, upon addition of excess zinc, HRV2 2A(pro) rapidly gained activity. Finally, the presence of the zinc chelator in the culture medium can protect HeLa cells from HRV infection.