Interband DNA of Drosophila melanogaster polytene chromosomes was studied using a novel approach based on the electron microscopic (EM) analysis of chromosome regions carrying DNA fragments of known molecular genetic composition, inserted by P element-mediated transformation. Insertion of such fragments predominantly into interbands makes it possible to clone interband DNA by constructing genomic libraries from transformed strains and probing them with the insert DNA. The transformed strain P[H-sp70:Adh](61C) has insertion in the 61C7-8 interband on the left arm of chromosome 3. This DNA consists of part of the hsp70 gene promoter fused to the coding region of the Adh gene, and is flanked on either side by P element sequences. We constructed a genomic library from DNA of this strain and isolated a clone containing the insert and the interband DNA. Subsequently the genomic library of wild-type strain was probed with a subclone composed of interband DNA only. We have thus isolated a clone containing the entire native interband. 1289 bp of interband DNA was sequenced and found to be AT-rich (53.4%) with numerous regions of overlapping direct and inverted repeats, regulatory sites, and two overlapping open reading frames (ORFs).