Flap endonuclease 1 (FEN1) is a structure-selective nuclease best known for its roles in the penultimate steps of Okazaki fragment maturation, long-patch base excision repair and ribonucleotide excision repair. To better understand the role of FEN1 in genome maintenance in yeast and mammals, FEN1 active site mutations (A159V and E160D) have been used as tools to dissect its involvement in DNA metabolic pathways. However, discrepancies concerning the biochemistry and molecular etiology of genomic instability when FEN1 function is altered exist. Here, a detailed biochemical and biophysical characterization of mouse FEN1 and mutants is presented. Kinetic measurements showed that the active site mutants A159V and E160D reduce the rates of hydrolysis under multiple- and single-turnover conditions on all substrates. Consistent with their dominant negative effects in heterozygotes, neither mutation affects the adoption of the substrate duplex arms in the bent conformation on the enzyme surface, although decreases in substrate binding affinity are observed. The ability of the mutants to induce the requisite local DNA conformational change near the scissile phosphate is adversely affected, suggesting that the ability to place the scissile phosphate optimally in the active site causes the reduction in rates of phosphate diester hydrolysis. Further analysis suggests that the A159V mutation causes the chemistry of phosphate diester hydrolysis to become rate-limiting, whereas the wild-type and E160D proteins are likely rate-limited by a conformational change. On the basis of these results, the proposed roles of FEN1 in genome maintenance derived from studies involving these mutations are reassessed.