Intracellular microtubule motor proteins may direct the motile properties and/or morphogenesis of the mitotic spindle (reviewed in ref. 3). The recent identification of kinesin-like proteins important for mitosis or meiosis indicates that kinesin-related proteins may play a universal role in eukaryotic cell division, but the precise function of such proteins in mitosis remains unknown. Here we use an in vitro assay for spindle assembly, derived from Xenopus egg extracts, to investigate the role of Eg5, a kinesin-like protein in Xenopus eggs. Eg5 is localized along spindle microtubules, and particularly enriched near spindle poles. Immunodepletion of Eg5 from egg extracts markedly reduces the extent of spindle formation in extracts, as does direct addition of anti-Eg5 antibodies. We also demonstrate that Eg5 is a plus-end-directed microtubule motor in vitro. Our results suggest a novel mechanism for the dynamic self-organization of spindle poles in mitosis.