Poxvirus DNA polymerases play a critical role in promoting virus recombination. To test if vaccinia polymerase (E9L) could mediate this effect by catalyzing the post-synaptic processing of recombinant joint molecules, we prepared substrates bearing a nick, a 3'-unpaired overhang, a 5' overhang, or both 3' and 5' overhangs. The sequence of the 5' overhang was also modified to permit or preclude branch migration across the joint site. These substrates were incubated with E9L, and the fate of the primer strand characterized under steady-state reaction conditions. E9L rapidly excises a mispaired 3' strand from a DNA duplex, producing a meta-stable nicked molecule that is a substrate for ligase. The reaction was not greatly affected by adding an unpaired 5' strand, but since such molecules cannot be processed into nicked intermediates, the 3'-ended strand continued to be subjected to exonucleolytic attack. Incorporating homology into the 5' overhang prevented this and permitted some strand assimilation, but such substrates also promoted strand-displacement DNA synthesis of a type predicted by the 1981 Moyer and Graves model for poxvirus replication. Single-strand annealing reactions are used by poxviruses to produce recombinant viruses and these data show that virus DNA polymerases can process DNA in such a manner as to both generate single-stranded substrates for such reactions and to facilitate the final processing of the reaction products.