The maintenance of a polarized cell surface requires vectorial transport of vesicles to the apical and the basolateral membrane domains. Transport of newly synthesized apical proteins and trans-cytosis from the basolateral to the apical surface have been demonstrated to depend on microtubules. In contrast, movement of membrane proteins to the basolateral surface has been claimed to occur by diffusion and to be microtubule- and actin-independent. We have re-examined the role of microtubules using a recently developed polarized transport assay in permeabilized Madin-Darby canine kidney cells. Here we report that both apical and basolateral transport is inhibited by nocodazole treatment. Transport to the basolateral surface was inhibited by immunodepletion of cytosolic kinesin. In contrast, apical transport involved both dynein and kinesin. Our data demonstrate that in epithelial cells, microtubule motors are involved in the movement of apical and basolateral vesicles. Moreover, we propose that the differential requirement for microtubule-based motors is related to the microtubule organization.