A specific cleavage by the reverse transcriptase-associated RNase H activity generates the RNA primer for plus strand DNA synthesis during reverse transcription. Previously, we used site-directed mutagenesis to define the sequence features of the polypurine tract (PPT) required for correct plus strand priming by the Moloney murine leukemia virus (M-MuLV) reverse transcriptase (Rattray, A. J., and Champoux, J. J. (1989) J. Mol. Biol. 208, 445-456). Although the sequences of human immunodeficiency virus type 1 (HIV-1) and M-MuLV diverge completely outside a 20-base region encompassing the PPT, within this region there are only three differences between the two viruses. Here we show that the HIV-1 reverse transcriptase will utilize the M-MuLV PPT as an origin for plus strand initiation in vitro. This finding enabled us to use the set of PPT mutants previously generated in M-MuLV, in conjunction with a small set of newly derived mutations within the HIV-1 PPT, to study plus strand priming by the HIV-1 reverse transcriptase. Despite the similarity between the two PPT regions, the sequence features important for positioning RNase H for the cleavage reaction that generates the plus strand primer are different for the two viruses. For M-MuLV, the -7A residue is a critical specificity determinant in the priming reaction, whereas for HIV-1, the -2G and -4G residues play key roles in determining the specificity of priming.