Annealing studies were performed on DNA fragments associated with rat and mouse liver interphase nuclear matrix and the metaphase scaffold of Chinese hamster DON cells. Matrix and scaffold bound DNA fragments, reassociated with an excess of total genomic DNA, displayed kinetics virtually identical with total nuclear DNA probes. Moreover, both the extent and kinetics of these hybridizations were independent of the matrix DNA fragment size (less than 350--5000 base pairs) and the method of nuclease digestion used in their preparation (DNase I, micrococcal nuclease or endogenous digestion). The repetitive DNA component of the matrix DNA was examined by reacting discrete sizes of matrix DNA fragments (less than 350--5000 base pairs) from mouse liver with a library of cloned repetitive sequence DNA fragments which included mouse major satellite sequences. Our results demonstrate that short DNA fragments anchored to the nuclear matrix contain these cloned sequences is similar proportion of total nuclear DNA and, when viewed in light of the annealing results, indicate that matrix DNA is not enriched in either repetitive or unique sequences. Furthermore, the matrix DNA fragments appear to contain the entire sequence complexity of the genome. Finally, we hybridized both matrix and total nuclear DNA fragments with cDNA to total nuclear polyadenylated RNA. The kinetics and extent of hybridization indicate that most, if not all, of the actively transcribed DNA sequences are present in similar concentrations. We conclude that in the overall organization of eukaryotic DNA within the nucleus, the repeating domains or loops which have been demonstrated by a number of investigators are not anchored at specific attachment sequences in interphase cells or during mitosis. These findings are discussed with regard to current concepts of eukaryotic DNA loop organization.