A 35-kDa polypeptide belonging to the high mobility group family of proteins was purified from the yeast Saccharomyces cerevisiae on the basis of its association with a DNA helicase activity. Amino acid sequence alignment suggests that this protein, Hmo1p, is related to the HMG1/2 class of chromatin-associated proteins. Consistent with this prediction, the Hmo1 protein immunolocalizes to the nucleus, binds single-stranded DNA, and unwinds DNA in the presence of eukaryotic DNA topoisomerase I. While the purified protein has no DNA helicase activity on its own, immunoprecipitation experiments confirm that Hmo1p associates with a 5' to 3' DNA helicase activity in nuclear extracts. The in vivo role of the protein was investigated by constructing an hmo1 deletion mutant. This strain has a severe growth defect, reduced plasmid stability, and chromatin that is hypersensitive to micrococcal nuclease digestion. Taken together, the data indicate that HMO1 is likely to be the homolog of HMG1/2 in higher cells and that it plays an important role in genome maintenance.