A homolog of the fission yeast cdc2-encoded protein kinase (p34) is a component of M phase promoting factor in Xenopus oocytes. The homologous kinase in human HeLa cells is maximally active during mitosis, suggesting a mitotic role in mammalian somatic cells. This has been directly investigated by microinjection of anti-p34 antibodies into serum-stimulated rat fibroblasts. DNA synthesis was unaffected but cell division was quantitatively blocked in injected cells. Injection of antibodies against p13suc1, a component of the p34 kinase complex, did not block mitosis but caused mitotic abnormalities resulting in cells containing multiple micronuclei in the subsequent interphase. p34 localized in the nucleus during interphase. During mitosis, a fraction tightly associated with centrosomes. p13 was more evenly distributed between the nucleus and cytoplasm. These observations demonstrate that cdc2 is a nuclear and centrosomal protein that is required for mitosis in mammalian cells.