We have isolated a murine cDNA, Mrad9, that is orthologous to the fission yeast rad9+ and human HRAD9 genes. Mrad9 encodes a 389 amino acid long, 42,032 Dalton protein that is 27% identical and 56% similar to Rad9p, and 82% identical and 88% similar to HRAD9, at the amino acid level. Expression of the Mrad9 cDNA in Schizosaccharomyces pombe rad9::ura4+ cells restores nearly wild-type levels of hydroxyurea resistance and early S phase checkpoint control to mutant fission yeast cell populations. However, UV resistance is only minimally restored, and mutant cells remain sensitive to gamma radiation. Mrad9 genomic DNA was isolated from a mouse 129/SvEv library. The Mrad9 gene was local ized to a 15-kbp genomic DNA fragment, and contains 10 exons separated by 9 introns. Northern blot analysis indicates that the gene is expressed in many different tissues of the adult mouse, but the mRNA is most abundant in the heart and present at very low levels in the liver. These studies demonstrate the existence of a murine orthologue of the fission yeast rad9+ gene and underscore at least the partial evolutionary conservation of rad9+-dependent checkpoint control mechanisms.