Gly-952 is a conserved residue in Saccharomyces cerevisiae DNA polymerase alpha (pol alpha) that is strictly required for catalytic activity and for genetic complementation of a pol alpha-deficient yeast strain. This study analyzes the role of Gly-952 by characterizing the biochemical properties of Gly-952 mutants. Analysis of the nucleotide incorporation specificity of pol alpha G952A showed that this mutant incorporates nucleotides with extraordinarily low fidelity. In a steady-state kinetic assay to measure nucleotide misincorporation, pol alpha G952A incorporated incorrect nucleotides more efficiently than correct nucleotides opposite template C, G, and T. The fidelity of the G952A mutant polymerase was highest at template A, where the ratio of incorporation of dCMP to dTMP was as high as 0.37. Correct nucleotide insertion was 500- to 3500-fold lower for G952A than for wild type pol alpha, with up to 22-fold increase in pyrimidine misincorporation. The Km for G952A pol alpha bound to mismatched termini T:T, T:C, C:A, and A:C was 71- to 460-fold lower than to a matched terminus. Furthermore, pol alpha G952A preferentially incorporated pyrimidine instead of dAMP opposite an abasic site, cis-syn cyclobutane di-thymine, or (6-4) di-thymine photoproduct. These data demonstrate that Gly-952 is a critical residue for catalytic efficiency and error prevention in S. cerevisiae pol alpha.