We previously reported that a mutant Rous sarcoma virus (RSV) with an alternate polypurine tract (PPT), DuckHepBFlipPPT, had unexpectedly high titers and that the PPT was miscleaved primarily at one position following a GA dinucleotide by the RNase H of reverse transcriptase (RT). This miscleavage resulted in a portion of the 3' end of the PPT (5'-ATGTA) being added to the end of U3 of the linear viral DNA. To better understand the RNase H cleavage by RSV RT, we made a number of mutations within the DuckHepBFlipPPT and in the sequences adjacent to the PPT. Deleting the entire ATGTA sequence from the DuckHepBFlipPPT increased the relative titer to wild-type levels, while point mutations within the ATGTA sequence reduced the relative titer but had minimal effects on the cleavage specificity. However, mutating a sequence 5' of ATGTA affected the relative titer of the virus and caused the RNase H of RSV RT to lose the ability to cleave the PPT specifically. In addition, although mutations in the conserved stretch of thymidine residues upstream of the PPT did not affect the relative titer or cleavage specificity, the mutation of some of the nucleotides immediately upstream of the PPT did affect the titer and cleavage specificity. Taken together, our studies show that the structure of the PPT in the context of the cognate RT, rather than a specific sequence, is important for the proper cleavage by RSV RT.