The exceptional cytology provided by polytene chromosomes has made Drosophila melanogaster a premier model for chromosome studies, but full exploitation of polytene cytology is impeded by the difficulty in preparing high-quality chromosome spreads. Here we describe use of high pressure to produce formaldehyde-fixed chromosome spreads, which upon light-microscopy examination reveal structural detail previously observed only in electron microscopy preparations. We demonstrate applications to immunofluorescence and in situ hybridization.