We describe a novel Xenopus plus end-directed kinesin-like protein (KLP), Xklp2, localized on centrosomes throughout the cell cycle and on spindle pole microtubules during metaphase. Using mitotic spindles assembled in Xenopus egg extracts and different recombinant GST-Xklp2 mutants, we show that this motor is targeted to spindle poles through its C-terminal domain. Xklp2-truncated polypeptides lacking the motor domain block centrosome separation and disrupt preassembled metaphase spindles. Antibodies directed against the tail of Xklp2 have a similar effect. These results show that Xklp2 protein is required for centrosome separation and maintenance of spindle bipolarity. This study is an example of the application of the dominant negative mutant effect on spindle assembly in Xenopus egg extracts, demonstrating the usefulness of this approach in probing the function of proteins in this system.