Katanin is a microtubule-severing protein that is concentrated at mitotic spindle poles but katanin's function in the mitotic spindle has not been previously reported. Inhibition of katanin with either of two dominant-negative proteins or a subunit-specific antibody prevented the redistribution of gamma-tubulin from the centrosome to the spindle in prometaphase CV-1 cells as assayed by immunofluorescence microscopy. Because gamma-tubulin complexes can bind to pre-existing microtubule minus ends, these results could be explained by a model in which the broad distribution of gamma-tubulin in the mitotic spindle is in part due to cytosolic gamma-tubulin ring complexes binding to microtubule minus ends generated by katanin-mediated microtubule severing. Because microtubules depolymerize at their ends, we hypothesized that a greater number of microtubule ends generated by severing in the spindle would result in an increased rate of spindle disassembly when polymerization is blocked with nocodazole. Indeed, katanin inhibition slowed the rate of spindle microtubule disassembly in the presence of nocodazole. However, katanin inhibition did not affect the rate of exchange between polymerized and unpolymerized tubulin as assayed by fluorescence recovery after photobleaching. These results support a model in which katanin activity regulates the number of microtubule ends in the spindle.