Many DNA polymerases select their natural substrates, deoxy- as opposed to ribonucleoside triphosphates, with a selectivity greater than 10000-fold. The function of a highly conserved residue, Tyr416, in the palm domain of the parental enzyme, an exo(-) derivative of RB69 DNA polymerase (gp43), a member of the pol alpha DNA polymerase family, was examined for its role in helping the polymerase discriminate between ribo-, dideoxyribo-, and deoxyribonucleoside triphosphates. The parental enzyme selected dNTPs vs rNTPs with about the same preference as dNTPs vs ddNTPs. Pre-steady-state kinetic analysis was carried out with the parental enzyme and two mutants, Y416A and Y416F. The Y416A mutant incorporated ribonucleotide residues much more efficiently than the parental enzyme, whereas the Y416F mutant was more permissive toward ddNTP vs rNTP utilization than either the Y416A mutant or the parental enzyme. We also found that both dCDP and rCDP inhibited dCTP incorporation by the Y416A mutant, while only dCDP but not rCDP inhibited dCTP incorporation by the parental enzyme and the Y416F mutant. The parental enzyme and the Y416A and Y416F mutants were all able to add araCTP (1-beta-D-arabinofuranosylcytosine-5'-triphosphate) to a primer but with reduced efficiency relative to dCTP. Based on our kinetic results, interpreted in the context of the crystal structure of the RB69 gp43 ternary complex, we suggest that sugar discrimination is provided mainly by the Tyr416 side chain which can sterically block the 2'-OH group of an incoming rNTP.