The compartmentalization of chromatin-bound RNA polymerases was examined with HeLa chromosomes fractionated according to their size in sucrose/glycerol gradients. There was a good correlation between the enrichment of nucleolus-organizing chromosomes, i.e., D and G group chromosomes, and the level of chromosome-associated RNA polymerase form I activity. A profile of DEAE-Sephadex chromatography of enzymes solubilized from chromosome fractions also supported the view that form I was associated with D and G group chromosomes. The amount of form I associated with unfractionated chromosomes was nearly identical to that of nuclei, indicating that this enzyme is probably quantitatively conserved even when chromatin undergoes extensive condensation. Since the chromatin-bound form I enzyme can be reactivated with heparin, it seems that this enzyme is in the initiated state, probably being bound to rDNA throughout the mitotic cycle. Thus, the absence of rRNA synthesis in mitosis is due to neither unavailability of enzyme to rDNA nor to the release of some factors necessary for transcriptional processes. Form II enzyme was associated uniformly with all chromosome fractions. Taken together, the present findings suggest that the intranuclear compartmentalization of RNA polymerases persists not only in interphase stages, but also in mitosis, during which most other nuclear proteins are released into the cytoplasm.