Bloom syndrome (BSx) is a rare autosomal-recessive chromosome-instability disorder manifested by a constellation of clinical features including a significant predisposition to early onset of neoplasia. BSx cells display cytogenetic abnormalities, the pathognomonic feature being an increased rate of spontaneous sister chromatid exchanges (SCEs), 10- to 15-fold more frequent than SCEs seen in control cells. Identification of the primary biochemical defect in BSx and its relationship to SCE frequency and neoplasia have been complicated by reports that BSx cell lines exhibit defects in the structure and/or activity of a number of different enzymes. The rare occurrence of the disorder and lack of informative families have precluded mapping of the primary defect by standard linkage analysis. We have utilized BSx cells as recipients for microcell-mediated chromosome transfer to map a locus that renders complementation of the elevated SCE phenotype. Studies with the BSx cell line GM08505 demonstrated a stable frequency of SCEs 10-fold higher than control values, offering a phenotype suitable for complementation studies. Transfer of different independent human chromosomes from somatic cell hybrids into BSx cells permitted identification of a single chromosome that dramatically reduced the SCE frequency to a level near that seen in control cells. Detailed characterization revealed this complementing element to be human chromosome 15.