NP95, which contains a ubiquitin-like domain, a cyclin A/E-Cdk2 phosphorylation site, a retinoblastoma (Rb) binding motif, and a ring finger domain, has been shown to be colocalized as foci with proliferating cell nuclear antigen in early and mid-S phase nuclei. We established Np95 nulligous embryonic stem cells by replacing the exons 2-7 of the Np95 gene with a neo cassette and by selecting out a spontaneously occurring homologous chromosome crossing over with a higher concentration of neomycin. Np95-null cells were more sensitive to x-rays, UV light, N-methyl-N"-nitro-N-nitrosoguanidine (MNNG), and hydroxyurea than embryonic stem wild type (Np95(+/+)) or heterozygously inactivated (Np95(+/-)) cells. Expression of transfected Np95 cDNA in Np95-null cells restored the resistance to x-rays, UV, MNNG, or hydroxyurea concurrently to a level similar to that of Np95(+/-) cells, although slightly below that of wild type (Np95(+/-)) cells. These findings suggest that NP95 plays a role in the repair of DNA damage incurred by these agents. The frequency of spontaneous sister chromatid exchange was significantly higher for Np95-null cells than for Np95(+/-) cells or Np95(+/-) cells (p < 0.001). We conclude that NP95 functions as a common component in the multiple response pathways against DNA damage and replication arrest and thereby contributes to genomic stability.