Basolateral to apical vesicular transcytosis in the hepatocyte is an essential pathway for the delivery of compounds from the sinusoidal blood to the bile and to traffic newly synthesized resident apical membrane proteins to their site of function at the canalicular membrane front. To characterize this pathway better, microtubules in a hepatocyte homogenate were polymerized by addition of taxol, and associated membrane-bound vesicles were isolated. This fraction was enriched in polymeric immunoglobulin A receptor and contained apical membrane proteins. Immunoelectron microscopy demonstrated that polymeric immunoglobulin A receptor was localized predominantly on vesicles ranging from 100 to 160 nm and that the multidrug resistance protein 2 and the bile salt export pump co-localized on these vesicles. The minus-ended microtubule motor, dynein, was highly enriched in the fraction, and its intermediate chain could be released effectively by incubation with 1 mM ATP or GTP. However, the association of the transcytotic vesicles with the microtubules was not sensitive to hydrolyzable or non-hydrolyzable nucleotides. This study characterizes a fraction of microtubule-associated vesicles from rat hepatocytes and demonstrates that several resident apical membrane transport proteins and the polymeric immunoglobulin A receptor traffic on the same vesicle.