The mechanisms used by murine polyomavirus for intracellular migration are yet to be clarified. In this work we selectively depolymerized microtubules or actin fibers and then studied the progression of polyomavirus infection in cultured cells. Our results demonstrate that microtubule depolymerization prevents polyomavirus migration toward the nucleus and from the nucleus to the cell surface, being also involved in viral release, while disruption of the actin microfilaments appears to have no detrimental effect on the virus ability to reach the nucleus. The ultrastructural observation of polyomavirus nonenveloped particles interacting with the free end and the lateral sides of microtubules together with the coimmunoprecipitation of tubulin and viral VP-1 further supports the idea that polyomavirus intracellular migration seems to be mediated by the interaction of polyomavirus major capsid protein VP-1 with tubulin.