The molecular basis that underlies the maintenance of polytene chromosome banding pattern remains unclear. To test the possibility that the decondensed state of interbands is provoked by the absence of nucleosomes, we have subjected chromatin from the previously defined 61C7/C8 interband to digestion with micrococcal nuclease. We have demonstrated that interband DNA forms nucleosomes both in salivary glands and in the bulk of larval tissues. This finding strongly suggests that the difference in compaction between DNA in polytene chromosome bands and interbands results from differences that appear at the higher levels of chromatin organization.