We studied whether interbands can be ectopically formed in Drosophila melanogaster polytene chromosomes. For comparative purposes, two types of P-element constructs were used. The first type was represented by P-element based insertions into compact bands. Sequences of these insertions or adjacent genomic sequences could be activated ectopically either by GAL4 or by dosage compensation machinery. In the second type, the DNA from transcriptionally silent interbands was positioned between the FRT sites, and was flanked by DNA sequences of genes that were also inactive in salivary glands. Electron microscopy analysis of salivary gland polytene chromosomes demonstrated that both types of constructs formed distinct, yet morphologically similar interbands. Notably, the second class of transposon insertions appeared in polytene chromosomes as two bands separated by one interband. Excision of interband material from such insertions resulted in fusion of newly appeared bands into a single band. We were able to confirm by molecular means that the DNA sequences in integrated constructs were intact, that chromatin organization of this DNA mimicked that of native interbands, and that it was accurately excised from the constructs by FLP. Thus, we demonstrate that transfer of interband DNA into a silent genetic environment does not compromise interband formation. Our results do not support the idea of the existence of distinct cytogenetic "band + interband" units, furthermore, they suggest the autonomy of the decompacted state of interbands.