We have combined nucleoside analog interference with chemical footprinting, thermal denaturation, NMR spectroscopy and biochemical studies to understand recognition of the polypurine tract (PPT) primer of the Saccharomyces cerevisiae LTR-retrotransposon Ty3 by its cognate reverse transcriptase (RT). Locked nucleic acid (LNA) analogs, which constrain sugar ring geometry, were introduced pairwise throughout the PPT (-) DNA template, while abasic tetrahydrofuran (THF) linkages, which lack the nucleobase but preserve the sugar-phosphate backbone, were introduced throughout the (-) strand DNA template and (+) strand RNA primer. Collectively, our data suggests that both the 5' and 3' portions of the PPT-containing RNA/DNA hybrid are sensitive to nucleoside analog substitution, while the intervening region can be modified without altering cleavage specificity. These two regions most likely correspond to portions of the PPT which make close contact with the Ty3 RT thumb subdomain and ribonuclease H (RNase H) catalytic center, respectively. Achieving similar phenotype with nucleoside analogs that have different effects on duplex geometry reveals structural features which are important mediators of Ty3 PPT recognition. Finally, introducing THF lesions around the scissile PPT/U3 junction indicates that template nucleobase -1 is dispensable for catalysis, while a primer nucleobase on either side of the junction is necessary.