Mitosis is characterized by condensation of chromatin, cessation of RNA transcription, and redistribution of nuclear proteins. We investigated the distribution of the hypo- and hyperphosphorylated forms of RNA polymerase II in mitotic cells from different cell lines by immunofluorescence. In interphase cells, the hyperphosphorylated RNA polymerase II (Pol IIO) is present in speckles and diffusely throughout the nucleoplasm. In prophase, when speckles disappear, Pol IIO concentrates at the surface of chromosomes and, in addition, localizes in small spots throughout the cytoplasm. The association of Pol IIO with the surface of chromosomes is visible until the chromosomes start to decondense during late anaphase/early telophase. In telophase cells, Pol IIO is absent in newly formed nuclei but present in the cytoplasm, while Pol IIO disappears nearly completely in late telophase cells. In early G1 cells, when cell nuclei increase in size, Pol IIO becomes present in the nucleus, first in small spots and later diffusely and in speckles. The hypophosphorylated form of RNA polymerase II (Pol IIA) is nearly absent in mitotic cells suggesting that Pol IIA is hyperphosphorylated at the onset of mitosis. Because Pol IIO, unlike Pol IIA, cannot assemble in transcription preinitiation complexes, the conversion of Pol IIA to Pol IIO and the lining of chromosomes with Pol IIO might be underlying a mechanism by which mitotic cells repress their transcriptional activity.