Poliovirus 2A(pro) is required for the inhibition of host cell protein synthesis and efficient viral replication. We investigated the role of 2A(pro) in regulating viral RNA stability, translation and replication in HeLa S10 reactions. The protease activity of 2A(pro) or its polyprotein precursors, 2AB or P2, was required to increase the stability of viral RNA and prolong translation. Since other viral proteins were not required for the observed effects of 2A(pro), it is likely that a cellular protein(s) modified by 2A(pro) mediated these effects on stability and translation. In addition, the protease activity of 2A(pro) stimulated negative-strand initiation by approximately five-fold but had no effect on positive-strand initiation. The 2A(pro) stimulation of negative-strand synthesis was independent of its effect on stability and translation. These findings further extend the previously known functions of protein 2A(pro) to include its role in increasing RNA stability, prolonging translation and stimulating negative-strand synthesis.