We have investigated proteasome localization in synchronized cells using polyclonal anti-proteasome antibodies. Proteasomes were localized in the nucleus and cytoplasm at all phases of the cycle, but changes in localization were observed which explain the different immunofluorescence patterns found in asynchronous cells. In the nucleus, the intensity of staining in early S phase was low and showed a punctate distribution which changed to a more diffuse and intense labeling during S to G1. In the cytoplasm, proteasomes were concentrated in the perinuclear region at G1 and at the start of S phase and gradually moved towards the periphery of the cell as the cell cycle progressed to G2. No cell cycle-dependent changes were detected in the rate of synthesis or level of proteasomes. An apparent colocalization of proteasomes with elements of the cytoskeleton mainly observed in G2 was investigated further in PtK2 cells. The overall distribution of proteasomes and cell cycle-dependent changes in PtK2 cells were similar to those in L-132 cells. Double-label immunofluorescence studies using anti-proteasome and anti-cytokeratin (TROMA-1) antibodies showed that proteasomes do colocalize with intermediate filaments of the cytokeratin type, mainly during G2. In mitosis, proteasomes were found by immunogold electron microscopy to be localized around the chromosomes in both PtK2 and L-132 cells. Cell cycle-dependent changes in the localization of proteasomes suggest that they may have a regulatory function related to the cell cycle, for example, in the degradation of proteins which control its progression.