Assembly of the mitotic spindle, the machinery responsible for chromosomal segregation, is regulated by Cdc2 kinase and requires mitotic chromatin. However, the molecular identity of the kinase substrate and chromatin factor is unknown. Here we have cloned a human complementary DNA encoding an evolutionarily conserved chromosomal protein of relative molecular mass 47,000 (M(r) 47K) which has three consensus motifs for Cdc2 kinase-mediated phosphorylation. The protein is phosphorylated only during mitosis and is associated with polypeptides having M(r)s of 31K, 67K and 200K. Mitotic arrest is induced by antisense messenger RNA or by affinity-purified autoantibody. In the arrested cells, the chromosomes remain unsegregated and the mitotic spindle is absent. We propose that the chromosomal protein is activated by phosphorylation at the interphase/mitosis transition by Cdc2 kinase, and that the protein, alone or as a complex, is a previously unidentified Cdc2 kinase substrate and chromatin factor necessary for spindle assembly.