Evolutionary rearrangements of pericentromeric heterochromatin among Drosophila melanogaster subgroup species have been investigated. Region-specific DNA library from Drosophila orena ovarian nurse cell chromocenter has been obtained by microdissection of polytene chromosomes. The probe was localized on ovarian nurse cells chromosomes of D. melanogaster subgroup species using fluorescent in situ hybridization. Sequences homologous to the sequences of the DNA-probe were found in chromocenter and pericentromeric regions of D. orena polytene chromosomes; and in all pericentromeric regions of other species with several exceptions. So, there was no labeling on one of the arms of D. simulans chromosome 2 but such sequences were present on telomere of D. erecta chromosome 3 and in regions adjacent to the brightly DAPI-stained heterochromatic blocks of D. yakuba, D. santomea and D. teissieri chromosomes 2 and 3. At S6 stage (secondary reticulate nucleus), the labeled chromatin in D. orena could be found mostly within a restricted area and no such chromatin could be detected throughout the rest of the nucleus. On the contrary, the labeled DNA was spread diffusely in the nuclei of other species at this stage.