Differential compaction of the interphase chromosomes is important for proper functioning of the eukaryotic genome. Such non-uniform compaction is most easily observed in Drosophila salivary gland polytene chromosomes as a reproducible banding pattern. Functional mechanisms underlying the establishment and maintenance of the banding pattern remain unclear but have been hypothesized to involve transcription and chromatin insulators. We tested functional properties of DNA fragments from several transcriptionally inert interband regions that behave as autonomous decompacted units of polytene chromosomes. Our results suggest that, in the absence of transcription, the decondensed state of interband regions does not depend on the presence of insulator elements but instead correlates with the presence of transcriptional enhancers.