The eukaryotic Y-box binding protein-1 (YB-1) functions in various biological processes, including transcriptional and translational control, DNA repair, drug resistance, and cell proliferation. To elucidate the physiological role of the YB-1 protein, we disrupted one allele of mouse YB-1 in embryonic stem (ES) cells. Northern blot analysis revealed that YB-1+/- ES cells with one intact allele contain approximately one-half the amount of mRNA detected in wild-type (YB-1+/+) cells. We further found that the protein level of YB-1+/- cells was reduced to approximately 50–60% compared with that of YB-1+/+ cells. However, no apparent growth difference was found between YB-1+/- and YB-1+/+ cells. YB-1+/- cells showed increased sensitivity to cisplatin and mitomycin C, but not to etoposide, X-ray or UV irradiation, as compared to YB-1+/+ cells. YB-1 may have the capacity to exert a protective role against cytotoxic effects of DNA damaging agents, and may be involved in certain aspects of drug resistance.