New methods are presented for the bulk isolation of metaphase chromosomes from HeLa cells, and an electron microscopic study of thin sections of these chromosomes is presented. The techniques for chromosome isolation were developed to utilize solution conditions that are as mild as possible, so that further biochemical and structural studies can be directly related to the in situ state of chromosomes. - Electron micrographs of thin sections of isolated HeLa metaphase chromosomes reveal the general organization of the nucleosome-containing fibers. Chromosomes in isolation buffer show a dense, relatively uniform distribution of material across the chromatids. Swollen chromosomes reveal the primary mode of organization of the fibers to be a radial distribution from the central axes of the chromatids. A significant proportion of the fibers could also be oriented longitudinally.