The mutant Chinese hamster cell line, EM9, is characterized by a high baseline sister-chromatid exchange (SCE) frequency, increased sensitivity to cell killing, and a defect in DNA strand-break repair. The molecular basis for this pleotrophic phenotype is not known. We examined, at the chromosomal level, the increased sensitivity of this mutant to incorporated BrdUrd. By varying the amount of BrdUrd in template DNA and measuring the frequency of SCEs and chromosomal aberrations, we demonstrated the enhanced sensitivity of EM9 to BrdUrd present in the template strand of DNA. Our results show that a 6-fold increase in SCEs occurs due to DNA replication over a BrdUrd-substituted template relative to a dThd-substituted template. With regard to aberration production in EM9, there is a significant enhancement of aberrations and a specific bias toward damage for the chromatid with Brdurd in the template strand. While these cells share some phenotypic properties with cells from patients with Bloom's syndrome, the genotypic similarities have not yet been established.