We have identified a region near the center of the dihydrofolate reductase gene dhfr in Chinese hamster ovary cells that is attached to nuclear scaffolds isolated by extraction with lithium diiodosalicylate. Detailed analysis presented here reveals the presence of only two closely linked sites in 35,000 base-pairs scanned that mediate attachment of the dhfr gene to the nuclear scaffold. Sequence analysis of one of the sites reveals a high A + T content, the presence of cleavage consensus sequences for topoisomerase II, and direct and inverted repeated sequence motifs that are localized to a small region of the attachment site. Attachment of these two regions to the nuclear scaffold is observed in wild-type, hemizygous, and amplified cell lines. Attachment is also retained in dhfr mutants isolated in our laboratory, in which chromosomal lesions have occurred directly adjacent to the scaffold-associated regions. These two regions are not bound to scaffolds prepared from isolated metaphase chromosomes, suggesting that attachment of the dhfr gene is lost during mitosis.