Werner syndrome (WS) is an autosomal recessive disorder characterized by genomic instability and the premature onset of a number of age-related diseases. The gene responsible for WS encodes a member of the RecQ-like subfamily of DNA helicases. Here we show that its murine homologue maps to murine chromosome 8 in a region syntenic with the human WRN gene. We have deleted a segment of this gene and created Wrn-deficient embryonic stem (ES) cells and WS mice. While displaying reduced embryonic survival, live-born WS mice otherwise appear normal during their first year of life. Nonetheless, although several DNA repair systems are apparently intact in homozygous WS ES cells, such cells display a higher mutation rate and are significantly more sensitive to topoisomerase inhibitors (especially camptothecin) than are wild-type ES cells. Furthermore, mouse embryo fibroblasts derived from homozygous WS embryos show premature loss of proliferative capacity. At the molecular level, wild-type, but not mutant, WS protein copurifies through a series of centrifugation and chromatography steps with a multiprotein DNA replication complex.