The catalytic efficiency of incorporation of deoxyribonucleotides by wild-type human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) was around 100-fold higher than for dideoxyribonucleotides, in Mg(2+)-catalyzed reactions, and more than 10,000-fold higher than for nucleotides having a 2'-hydroxyl group in Mg(2+)- and Mn(2+)-catalyzed reactions. Mutant RTs with nonconservative substitutions affecting Tyr-115 (Y115V, Y115A, and Y115G) showed a dramatic reduction in their ability to discriminate against ribonucleotides in the presence of both cations. However, selectivity of deoxyribonucleotides versus ribonucleotides was not affected in mutants Y115W and F160W. The substitution of Tyr-115 with Val or Gly had no effect on discrimination against dideoxyribonucleotides, but these mutants were less efficient than the wild-type RT in discriminating against cordycepin 5'-triphosphate. We also show that Tyr-115 is involved in fidelity of DNA synthesis, but substitutions at this position have different effects depending on the metal cofactor used in the assays. In Mg(2+)-catalyzed reactions, removal of the side chain of Tyr-115 reduced misinsertion and mispair extension fidelity, while opposite effects were observed in Mn(2+)-catalyzed reactions. Our results indicate that the aromatic side chain of Tyr-115 plays a role as a "steric gate" preventing the incorporation of nucleotides with a 2'-hydroxyl group in a cation-independent manner, while its influence on fidelity could be modulated by Mg(2+) or Mn(2+).