Proteins tightly bound to the DNA of mouse L cells were examined both in the regions of DNA attachment to the protein skeleton and in the DNA loops. The interphase nuclear matrix and the metaphase chromosomal scaffold, containing from 2 to 15% of the original DNA, as well as the material released from the skeleton were isolated by mild nuclease treatment. At least six proteins resistant to sarcosyl-Cs2SO4 treatment are associated exclusively with skeleton-attached DNA both in interphase nuclei and metaphase chromosomes. Two (p52 and p60) can be dissociated from the complex by mercapto-ethanol-sarcosyl treatment. Both skeleton-attached and released DNA fractions contain a group of tightly bound low molecular weight polypeptides consisting of a major p18 band and one or two minor bands. They seem to be randomly distributed throughout the whole DNA loop. Some of the specific proteins that are tightly associated with DNA at the site of its interaction with the skeleton may directly be responsible for DNA attachment to the skeleton.