Due to the striking correlation between the distribution of transcriptionally active subdivisions of the polytene chromosomes and Z-DNA, we have addressed the question of whether the Z-DNA configuration exists in native, transcriptionally active chromosomes of Drosophila hydei prepared without interference by procedures known to induce the B to Z conformation. Our experiments indicate that Z-DNA forms are present in a specific set of sites on the native chromosomes. They occur on interbands and other subdivisions of dispersed DNA, but there is no correlation between the amount of Z-DNA detected and DNA compaction. The results suggest, moreover, that Z-DNA forms are restricted to specific genes, because various subdivisions induced to transcription in puffs show different patterns of Z-DNA. We show, in addition, that removal of chromosomal proteins by proteinase K has a strong influence on the level of anti-Z-DNA reactivity.