The physical association of mammalian excision repair enzymes with DNA was examined as a function of cell proliferation. The molecular weight distribution of two nuclear base excision repair enzymes, the uracil DNA glycosylase and the hypoxanthine DNA glycosylase, were examined by sucrose step gradient analysis. The sedimentation of DNA polymerase activity as well as the distribution of parental and replicating DNA were determined simultaneously. In confluent BHK-21 fibroblasts, basal levels of both DNA glycosylases, DNA polymerase beta, and parental DNA sedimented to the 20%/40% sucrose border. In proliferating BHK-21 cells, induced levels of both DNA glycosylases, DNA polymerase alpha, and replicating DNA sedimented to the 40%/50% sucrose border. The physical association of the repair enzymes with DNA was demonstrated by detergent treatment and by DNase digestion. As defined by [35S] methionine pulse labeling analysis, newly synthesized DNA repair enzymes were localized with either parental or replicating DNA. These results suggested that the physical association of mammalian DNA repair enzymes with nuclear DNA was dependent on the proliferative state of the cell.