Ribonuclease H (EC 3.1.4.34) has been used to remove selectively much of the 3'-terminal poly(adenylic acid) [poly(A)] from poliovirus RNA by treating the RNA with the enzyme in the presence of poly(dT). Over 80% of the poly(A) could be removed and the residuum was found as oligo(A) stretches attached to many or all of the viral RNA molecules. Reduction of the size of the poly(A) markedly decreased the specific infectivity of poliovirus RNA, indicating that poly(A) is necessary to the infectivity of the RNA. The virions in plaques deriving from infection with treated RNA have a normal amount and size of poly(A), indicating that mechanisms exist in infected cells to regenerate normal length poly(A) from truncated poly(A).