The primary processing of the poliovirus polyprotein is catalyzed by 2A protease (2Apro) which cleaves at the 1D/2A junction in a very rapid cotranslational reaction. In addition, 2Apro also indirectly induces cleavage of the p220 component of eIF-4F, which results in selective inhibition of host protein synthesis. Earlier studies have indicated that 2Apro is related to 3C protease (3Cpro) and is structurally similar to trypsin-like serine proteases with the substitution of Cys109 as the nucleophile. We noticed that 2Apro of enteroviruses and rhinoviruses contains a specific motif of Cys55-Xaa-Cys57-Xaan-Cys115-Xaa-His117 which is absolutely conserved, but which is not found in viral 3Cpro or known cellular serine proteases. To better understand the specific roles these conserved cysteine and histidine residues played in the structure/function of 2Apro, we constructed a series of 2Apro mutants by site-specific mutagenesis and analyzed the mutant enzymes with respect to their biochemical properties. Conservative amino acid replacements at Cys55, Cys57, Cys115, or His117 resulted, in each case, in a complete loss of both in cis and in trans activities of 2Apro. To determine the function of these residues, we examined the biochemical/structural features of 2Apro expressed in a cell-free rabbit reticulocyte lysate system. Gel mobility shift and chemical modification data suggest that these cysteine residues do not form intra-molecular disulfide linkages as a structural feature of 2Apro. However, studies with metal chelators did not eliminate the possibility that 2Apro contains a metal-binding ligand. Finally, our results suggest that these conserved cysteine and histidine residues, including Cys55, Cys57, Cys115, and His117, are critical in maintaining the active conformation of 2Apro structure and essential in supporting the catalytic activity of 2Apro.