A conserved purine-rich motif located near the 3' end of retroviral genomes is involved in the initiation of plus-strand DNA synthesis. We mutated sequences both within and flanking the Moloney murine leukemia virus polypurine tract (PPT) and determined the effects of these alterations on viral DNA synthesis and replication. Our results demonstrated that both changes in highly conserved PPT positions and a mutation that left only the cleavage-proximal half of the PPT intact led to delayed replication and reduced the colony-forming titer of replication defective retroviral vectors. A mutation that altered the cleavage proximal half of the PPT and certain 3' untranslated region mutations upstream of the PPT were incompatible with or severely impaired viral replication. To distinguish defects in plus-strand priming from other replication defects and to assess the relative use of mutant and wild-type PPTs, we examined plus-strand priming from an ectopic, secondary PPT inserted in U3. The results demonstrated that the analyzed mutations within the PPT primarily affected plus-strand priming whereas mutations upstream of the PPT appeared to affect both plus-strand priming and other stages of viral replication.