The Ultrabithorax gene is required to specify the third thoracic and first abdominal segments of Drosophila melanogaster. Mutations in the bithoraxoid region, a 40 kb DNA stretch upstream of the Ultrabithorax promoter, affect cis-regulatory elements of the Ultrabithorax gene. We now have identified specific sites in the bithoraxoid region that exhibit S1 nuclease sensitivity in vitro. These sites are not scattered along the DNA but are grouped instead in specific domains. Some of these S1-sensitive sites correlate with known breakpoint or insertional mutations. Others correspond to putative binding sites for transcription factors. The results suggest that unusual secondary structure might be important in chromosomal translocation within regulatory sequences of the Ultrabithorax product or its transcriptional regulation.