A mild enzymic procedure for fractionating nuclei was based on a combination of light-scattering properties and electron microscopy in order to monitor the structural integrity of rat liver nuclei and to establish the gentlest conditions possible for their disruption. Incubation of nuclei with as little as 0.1 unit of micrococcal nuclease per ml for 60 seconds at 20 to 29 °C, followed by EGTA, caused their total disruption with minimal perturbation of chromatin or transcriptional characteristics. A simple two-step differential centrifugation resolved the gently disrupted nuclei into three mechanically unsheared fractions. One of these (fraction P2) consisted of aggregates of 6 to 30 covalently linked nucleosomes, each containing about 200 base-pairs of DNA, and which are here termed polynucleosomes. This fraction represented about 10% of nuclear DNA (200 to 6000 base-pairs), whose properties corresponded to euchromatin prepared by other methods. Since there is virtually no reinitiation of RNA synthesis in vitro by isolated nuclei or subnuclear preparations, endogenous RNA polymerase activities represent the elongation of RNA chains that were initiated in vivo. When the distribution of free (inactive in the absence of exogenous template) and endogenous template-engaged RNA polymerases I(A) and II(B) was monitored, the latter as an index of transcriptional complexes that existed in the intact nucleus, mild nuclease digestion was found not to alter the autonomous transcriptional characteristics of the disrupted nuclei. Over 85% of the template-engaged RNA polymerase II was recovered with polynucleosomes. Further nuclease digestion of this fraction showed that a minimum of six nucleosomes/unit was necessary for retaining the enzyme on its template. Polynucleosomes can therefore be considered as the basic structural units of chromatin which are selectively released by extremely mild enzymic treatment of whole nuclei from the transcriptionally active compartment and are capable of continuing in vitro the elongation of RNA chains initiated in vivo.