The VP55 (catalytic) subunit of vaccinia virus heterodimeric poly(A) polymerase (PAP) contacts 31-40 nucleotide segments of RNA in a uridylate-dependent manner, and effects the rapid, processive addition of a 30 nt oligo(A) tail. Here, the minimum size of uridylate-containing RNA required for stable VP55 interaction was refined to 33-34 nt. VP55 binding experiments using a set of sixteen 34 nt DNA-RNA chimeras, each containing a differently positioned tetra-uridylate cluster within an oligo(dC) background, indicated that the protein contacts uridylates at two positions within the oligonucleotide. Combination of two optimally positioned tetra-uridylate clusters into a single oligonucleotide fully restored the properties of an optimal substrate, rU(34), in VP55 binding and salt-resistant polyadenylylation. The positions of the two uridylate interaction sites, similar to 10 and similar to 25 nt from the oligonucleotide 3' OH, were confirmed using a selection scheme employing dC-rU oligonucleotide chimera pools. These and additional data suggest a mechanism for polymerase translocation with respect to RNA comparable with inchworming models of transcriptional elongation. In selection experiments incorporating the PAP-associated processivity factor VP39, the latter was shown to replace the 3' OH-distal uridylate contact site with one similar to 10 nt further upstream.