To better understand the functions and fidelity of DNA polymerase epsilon (Pol epsilon), we report here on the fidelity of yeast Pol epsilon mutants with leucine, tryptophan or phenylalanine replacing Met644. The Met644 side chain interacts with an invariant tyrosine that contacts the sugar of the incoming dNTP. M644W and M644L Pol epsilon synthesize DNA with high fidelity, but M644F Pol epsilon has reduced fidelity resulting from strongly increased misinsertion rates. When Msh6-dependent repair of replication errors is defective, the mutation rate of a pol2-M644F strain is 16-fold higher than that of a strain with wild-type Pol epsilon. In conjunction with earlier studies of low-fidelity mutants with replacements for the homologous amino acid in yeast Pol alpha (L868M/F) and Pol delta (L612M), these data indicate that the active site location occupied by Met644 in Pol epsilon is a key determinant of replication fidelity by all three B family replicative polymerases. Interestingly, error specificity of M644F Pol epsilon is distinct from that of L868M/F Pol alpha or L612M Pol delta, implying that each polymerase has different active site geometry, and suggesting that these polymerase alleles may generate distinctive mutational signatures for probing functions in vivo.