The Ku protein is a DNA-binding nuclear protein complex composed of 86 kDa and 70 kDa subunits. Recently, in vitro studies suggested a role of the Ku protein in the activation of gene transcription. We studied the expression of these proteins during cell proliferation by Northern blot hybridizations using specific cDNA probes and by immunofluorescence and immunoblot analysis using specific monoclonal antibodies. The genes coding for both subunits were activated during late G1-phase in the transition of human PHA-stimulated lymphocytes from quiescent (G0 phase) to proliferative (S phase) state. These genes were inactivated when human leukemia cells HL60 were differentiated into monocytes upon treatment with the phorbol ester TPA. Changes at the protein level were significantly smaller than changes at the mRNA levels in both cell systems, suggesting a high stability of the Ku protein. Immunofluorescence studies demonstrated nucleolar as well as nuclear localization of the Ku protein in quiescent lymphocytes and during the early G1-phase; during the late G1, S and G2 phases, the Ku protein was only localized in discrete structures in the nucleoplasm. These results demonstrate that the gene expression for the Ku protein is associated with the proliferative state of the cells and that the nucleolar localization of the Ku protein is cell-cycle-dependent.