Survivin is a member of the inhibitor of apoptosis gene family that has been implicated in both apoptosis inhibition and regulation of mitosis. However, the subcellular distribution of survivin has been controversial and variously described as a microtubule-associated protein or chromosomal passenger protein. Here, we show that antibodies directed to the survivin sequence Ala³-Ile¹⁹ exclusively recognized a nuclear pool of survivin that segregated with nucleoplasmic proteins, but not with outer nuclear matrix or nuclear matrix proteins. By immunofluorescence,nuclear survivin localized to kinetochores of metaphase chromosomes, and to the central spindle midzone at anaphase. However, antibodies to Cys⁵⁷-Trp⁶⁷ identified a cytosolic pool of survivin,which associated with interphase microtubules, centrosomes, spindle poles and mitotic spindle microtubules at metaphase and anaphase. Polyclonal antibodies recognizing survivin epitopes Ala³-Ile¹⁹,Met³⁸-Thr⁴⁸, Pro⁴⁷-Phe⁵⁸ and Cys⁵⁷-Trp⁶⁷ identified both survivin pools within the same mitotic cell. A ratio of ∼1:6 for nuclear versus cytosolic survivin was obtained by quantitative subcellular fractionation. In synchronized cultures, cytosolic survivin abruptly increased at mitosis, physically associated with p34^cdc2, and was phosphorylated by p34^cdc2 on Thr³⁴, in vivo. By contrast, nuclear survivin began to accumulate in S phase, was not complexed with p34^cdc2 and was not phosphorylated on Thr³⁴. Intracellular loading of a polyclonal antibody to survivin caused microtubule defects and resulted in formation of multipolar mitotic spindles, but did not interfere with cytokinesis. These data demonstrate that although both reported localizations of survivin exist in mitotic cells, the preponderant survivin pool is associated with microtubules and participates in the assembly of a bipolar mitotic spindle.