The 2B proteins of coxsackievirus and poliovirus (PV) share significant structural similarity and exhibit similar biochemical activities, namely inhibition of protein secretion and modification of membrane permeability. Both proteins contain two hydrophobic domains in the carboxy-terminal two-thirds of their sequence, of which one has the potential to form a cationic amphipathic alpha-helix. To gain more insight into the structural requirements of enterovirus protein 2B for its functioning in viral RNA replication, a chimeric cDNA approach was used. Chimeric coxsackie B3 virus (CBV3) genomes were constructed that expressed either the entire PV 2B protein or hybrid proteins in which specific segments of CBV3 2B were substituted by their corresponding PV counterparts. In vitro synthesis and processing of the chimeric polyproteins showed no abnormalities. CBV3 genomes carrying the entire PV 2B gene failed to replicate. A chimeric genome that expressed a hybrid 2B protein consisting of the amino-terminal one-third of PV and the remainder of CBV3 yielded viable viruses. In contrast, a 2B protein consisting of the amino-terminal one-third of CBV3 and the remainder of PV failed to drive replication. These data imply that a sequence-specific interaction with another viral protein is required to drive RNA replication and suggest that the proposed sites of contact reside in the carboxy-terminal two-thirds of 2B. Hybrid genomes in which either the amphipathic alpha-helix or the other hydrophobic domain was replaced failed to replicate. The potential contribution of these domains to the structure and functioning of protein 2B are discussed.