Mitosin is a novel 350-kDa nuclear phosphoprotein that dramatically relocates from the evenly nuclear distribution in S phase to the centromere/kinetochore and mitotic apparatus in M phase. The dynamic relocalization of mitosin is accompanied by the phosphorylation of itself, suggesting that mitosin plays a role in mitotic progression. The molecular basis of nuclear localization and targeting of mitosin to the centromere/kinetochore were characterized using a set of epitope-tagged deletion mutants. The data indicate that the extreme C terminus (amino acids 2,487-3,113) of mitosin has both an independent centromere/kinetochore targeting domain and an unusually spaced bipartite nuclear localization signal. Moreover, the same centromere/kinetochore targeting domain was shown to be essential for the ability of mitosin to bind to itself or other putative mitosin-associated proteins through use of the yeast two-hybrid system. These results suggest that the C terminus of the mitosin is essential for its role in influencing cell cycle progression.