B10 is an integral glycoprotein of the plasma membrane that is exclusively localized to the canalicular (apical) domain in normal rat hepatocytes but may be expressed on the basolateral (sinusoidal and lateral) membrane in pathophysiological situations. To understand how B10 may be localized to the basolateral surface, we studied the biosynthesis and transport of this apical protein. In vivo pulse-chase experiments, followed by subcellular fractionation of the liver and immunoprecipitation, showed that B10 is first synthesized as a high-mannose form of 123 kD and then matured to a complex glycosylated form of 130 kD, which peaks in the Golgi apparatus after 15 min of chase and reaches the plasma membrane with a half-time of 30 to 45 min. Analysis of the protein in plasma membrane domain fractions showed that most of the newly synthesized molecule was localized in basolateral fractions after 30 min of chase and subsequently appeared in apical fractions. After 90 min of chase, most of the radiolabeled protein had reached its steady-state apical distribution. The same experiments performed in the perfused rat liver, in which the chase can be improved, gave similar results, except that the apical distribution of the radioactive molecule was attained more quickly. Thus B10, like all apical plasma membrane proteins studied so far in hepatocytes, is first transported to the basolateral surface and then reaches the membrane of the bile canaliculi. Alterations of the transcytotic step from the basolateral to the apical surfaces may result in abnormal basolateral localization.