DNA was isolated from chromosomes or nuclei scraped off slides after each step of the C-banding procedure. Analysis by buoyant density-gradient centrifuga-tion and digestion with nucleases specific for single-stranded nucleic acids showed: 80 % or more of the DNA remained double-stranded after fixation to slides and 70 % or more was single-stranded after the slides were treated with alkali, whether the slides were annealed afterwards or not. Furthermore, highly repeated DNAs remained single-stranded after annealing following denaturation. Since the annealing of slides after denaturation does not effect the reassociation of either bulk or highly repeated DNA sequences, C-banding apparently results from DNA-protein interactions rather than preferential strand reassociation of highly repeated DNAs.