The mechanism of encapsidation of the RNA genome of poliovirus and other picornaviruses is unknown. To test whether any of the putative assembly intermediates of poliovirus could interact directly with the poliovirus RNA genome, poliovirus RNA was attached to magnetic streptavidin beads and incubated with partially purified extracts containing 35S-labeled 14S pentamer and 75S empty-capsid subviral particles from infected cells. The amount of labeled protein bound to the beads was monitored, thus testing the RNA-binding activities of only the labeled viral proteins in the preparations. In this assay, nonspecific RNA-binding activity was displayed by the 14S pentameric particles and mature virons. 75S empty capsids displayed no propensity to associate with RNA. 14S pentamers were demonstrated to form rapidly sedimenting complexes and to undergo a conformational alteration upon RNA binding. These findings are consistent with a direct role for the 14S pentameric particles in RNA packaging during poliovirus morphogenesis.