The precise relationship between the length of the 3' poly(A) tail and the replication and infectivity of poliovirus RNA was examined in this study. With both poly(A)(11) and poly(A)(12) RNAs, negative-strand synthesis was 1-3% of the level observed with poly(A)(80) RNA. In contrast, increasing the length of the poly(A) tail from (A)(12) to (A)(13) resulted in about a ten-fold increase in negative-strand synthesis. This increase continued with each successive increase in poly(A) tail length. With poly(A)(20) RNA, RNA synthesis approached the level observed with poly(A)(80) RNA. A similar relationship was observed between poly(A) tail length and the infectivity of the viral RNA. A replication model is described which suggests that viral RNA replication is dependent on a poly(A) tail that is long enough to bind poly(A) binding protein and to act as a template for VPg uridylylation and negative-strand initiation.