Poliovirus proteinase 2A(pro) is an essential enzyme involved in cleavages of viral and cellular proteins during the infectious cycle. Evidence has been obtained that 2A(pro) is also involved in genome replication. All enteroviruses have a negatively charged cluster of amino acids at their C terminus (E(E)/(D)(E)/(D)AMEQ-NH(2)), a common motif suggesting function. When aligned with enterovirus sequences, the 2A(pro) proteinase of human rhinovirus type 2 (HRV2) has a shorter C terminus (EE.Q:-NH(2)) and, indeed, the HRV2 2A(pro) cannot substitute for poliovirus 2A(pro) to yield a viable chimeric virus. Here evidence is provided that the C-terminal cluster of amino acids plays an unknown role in poliovirus genome replication. Deletion of the EEAME sequence from poliovirus 2A(pro) is lethal without significantly influencing proteinase function. On the other hand, addition of EAME to HRV2 2A(pro), yielding a C terminus of this enzyme of EEEAMEQ:, stimulated RNA replication of a poliovirus/HRV2 chimera 100-fold. The novel role of the C-terminal sequence motif is manifested at the level of protein function, since silent mutations in its coding region had no effect on virus proliferation. Poliovirus type 1 Mahoney 2A(pro) could be provided in trans to rescue the lethal deletion EEAME in the poliovirus variant. Encapsidation studies left open the question of whether the C terminus of poliovirus 2A(pro) is involved in particle formation. It is concluded that the C terminus of poliovirus 2A(pro) is an essential domain for viral RNA replication but is not essential for proteolytic processing.