The REV3 gene encodes the catalytic subunit of DNA polymerase (pol) ζ, which can replicate past certain types of DNA lesions . Saccharomyces cerevisiae rev3 mutants are viable and have lower rates of spontaneous and DNA-damage-induced mutagenesis . Reduction in the level of Rev31, the presumed catalytic subunit of mammalian pol ζ, decreased damage-induced mutagenesis in human cell lines . To study the function of mammalian Rev31, we inactivated the gene in mice. Two exons containing conserved DNA polymerase motifs were replaced by a cassette encoding G418 resistance and β-galactosidase, under the control of the Rev3l promoter. Surprisingly, disruption of Rev3l caused mid-gestation embryonic lethality, with the frequency of Rev3l(-/-) embryos declining markedly between 9.5 and 12.5 days post coitum (dpc). Rev3l(-/-) embryos were smaller than their heterozygous littermates and showed retarded development. Tissues in many areas were disorganised, with significantly reduced cell density. Rev3l expression, traced by β-galactosidase staining, was first detected during early somitogenesis and gradually expanded to other tissues of mesodermal origin, including extraembryonic membranes. Embryonic death coincided with the period of more widely distributed Rev3l expression. The data demonstrate an essential function for routine Rev31 and suggest that bypass of specific types of DNA lesions by pol ζ is essential for cell viability during embryonic development in mammals.