The Xenopus laevis aurora/Ip11p-related kinase pEg2 is required for centrosome separation, which is a prerequisite for bipolar mitotic spindle formation. Here, we report that the inhibition of pEg2 by addition of either an inactive kinase or a monoclonal antibody destabilizes bipolar spindles previously assembled in Xenopus egg extracts. The bipolar spindles collapse to form structures such as microtubule asters with chromosome rosettes, monopolar spindles, and multipolar spindles. In collapsed spindles, chromosomes remain attached to the microtubules plus ends. The destabilization of the bipolar spindle is reminiscent of the destabilization observed after inhibition of cross-linking activities which maintain parallel and anti-parallel microtubules linked together. We have previously reported that pEg2 phosphorylates the kinesin-related protein XlEg5 which is involved in centrosome separation but which was also reported to be involved in spindle stability. The collapse of the bipolar spindle observed after inhibition of pEg2 suggests that the kinase might regulate the cross-linking activity of XlEg5. We do not exclude the possibility that pEg2 also regulates other microtubule-based motor proteins involved in bipolar spindle stability. To our knowledge, this is the first evidence that aurora/Ip11p-related kinase activity actually participates not only in mitotic spindle formation by regulating centrosome separation but also in mitotic spindle stabilization.