Abstract Flap endonuclease 1 (FEN1) processes Okazaki fragments in lagging strand DNA synthesis, and FEN1 is involved in several DNA repair pathways. The interaction of FEN1 with the proliferating cell nuclear antigen (PCNA) processivity factor is central to the function of FEN1 in both DNA replication and repair. Here we present two gene-targeted mice with mutations in FEN1. The first mutant mouse carries a single amino acid point mutation in the active site of the nuclease domain of FEN1 (Fen1E160D/E160D), and the second mutant mouse contains two amino acid substitutions in the highly conserved PCNA interaction domain of FEN1 (Fen1ΔPCNA/ΔPCNA). Fen1E160D/E160D mice develop a considerably elevated incidence of B-cell lymphomas beginning at 6 months of age, particularly in females. By 16 months of age, more than 90% of the Fen1E160D/E160D females have tumors, primarily lymphomas. By contrast, Fen1ΔPCNA/ΔPCNA mouse embryos show extensive apoptosis in the forebrain and vertebrae area and die around stage E9.5 to E11.5. [Cancer Res 2008;68(12):4571–8]