We have been able to map specific DNA fragments at the bases of chromatin loops with the help of a novel extraction procedure by using lithium-3',5'-diiodosalicylate. One such scaffold-attached region (SAR) is found in the non-transcribed spacer in each repeat of the histone gene cluster, on a 657 base pair (b.p.) restriction fragment. Exonuclease III digestion has localized two protein-binding domains on the SAR of the histone cluster. Each covers approximately 200 b.p. and they are separated by a nuclease-accessible region of about 100 b.p. These domains are rich in sequences closely related to the topoisomerase II cleavage consensus. We have studied the scaffold association of three developmentally regulated genes of Drosophila melanogaster: alcohol dehydrogenase (Adh), the homoeotic gene fushi tarazu (ftz) and Sgs-4, a gene encoding one of the glue proteins secreted by third-instar larvae. We find regions attached to the nuclear scaffold (SARS) both 5' and 3' of all three genes, defining small domains ranging from 4.5 to 13 kilobases. In the case of Adh, a gene with two promoters, we find two upstream and two downstream SARS. Those 5' of the gene co-map with regulatory regions for the adult and the larval transcripts, respectively. For Sgs-4, the 5' SAR covers 866 b.p. immediately upstream of the transcript, and encompasses the 200 b.p. regulatory region defined by two deletion mutants that produce little or no Sgs-4 protein. In ftz the 5' SAR is found 4.8 kilobases upstream of the start of transcription within a 2.5 kilobase element required for a high level of ftz expression in the early embryo. Sequence analysis of five upstream SARS reveals clusters of sequences closely related to the cleavage consensus of topoisomerase II. In addition, they contain multiple copies of two sequence motifs: a specific 10 b.p. A-rich sequence, and another 10 b.p. T-rich stretch. In conclusion, the intimate association of the SAR with the upstream/enhancer elements, the presence of clustered sequences highly homologous to the topoisomerase II cleavage consensus, and the localization of topoisomerase II in the scaffold, suggest a structure-function relation between chromosome organization and gene expression.