Recently, we reported that the monoclonal antibody specific for human DNA topoisomerase II␣, Ki-S1, stains not only the nuclei of human A431 cells but also extranuclear structures suggestive of centrosomes (Meyer, K. N., Kjeldsen, E., Straub, T., Knudsen, B. K., Kikuchi, A., Hickson, I. D., Kreipe, H., and Boege, F. (1997) J. Cell Biol. 136, 775-788). Here, we confirm colocalization of Ki-S1 with the centrosomal marker ␥-tubulin. In addition, we show labeling of centrosomes by peptide antibodies against the N and C termini of human topoisomerase II␣. Probing Western blots of isolated centrosomes with topoisomerase II␣ antibodies, we demonstrate a protein band of 170 kDa. Moreover, isolated centrosomes exhibited DNA decatenation and relaxation activity correlated to the amount of topoisomerase II␣ protein in the same way as seen in the pure recombinant enzyme. Topoisomerase II␣ epitopes could not be removed from centrosomes by salt extraction, DNase treatment, or RNase treatment, procedures that completely removed the enzyme from nuclei. Taken together, these observations suggest that active topoisomerase II␣ is bound tightly to the centrosome in a DNA-independent manner. Because such centrosomal topoisomerase II␣ was also present in quiescent lymphocytes devoid of topoisomerase II␣ in the nuclei, we assume that it might be a long-lived storage form.We have observed (1, 2) that the monoclonal antibody Ki-S1 directed against DNA topoisomerase II␣ (3) labels not only the cell nuclei (a long-standing observation) but also labels small globular structures located at the poles of mitotic spindles (a new finding). Ki-S1 labeling of extranuclear globules was reproducible in two human cell lines, whereas a similar observation was not made with antibodies directed against DNA topoisomerase I or II. We assumed that these Ki-S1-positive extranuclear structures would most likely be centrosomes. However, they could just as well be chromosome fragments or micronuclei, or the staining could be due to a spurious crossreaction of the Ki-S1 antibody with some centrosomal protein other than topoisomerase II␣. However, if such artifacts were excluded, our observation might indicate topoisomerase II␣ as a possible component of centrosomes. We found our chance observation interesting enough to be followed up. Here, we carried out immunocytochemical colocalization studies combining antibodies against ␥-tubulin (an established centrosomal marker) with antibodies against several distinct epitopes of DNA topoisomerase II␣. We corroborated such studies by biochemical analysis of isolated centrosomes. Our results confirm that active DNA topoisomerase II␣ is associated with centrosomes.