Liver plasma membrane ecto-ATPase activity is largely restricted to the bile canalicular membrane. To determine whether a transport process is also selectively present on this membrane surface to reclaim adenosine derived from the intracanalicular degradation of ATP, the characteristics of hepatic nucleoside transport were examined in canalicular (cLPM) and basolateral (blLPM) rat liver plasma membrane vesicles. In the presence of the adenosine deaminase inhibitor, deoxycoformycin, an inwardly directed Na+ gradient markedly stimulated [3H]adenosine uptake in cLPM vesicles. Canalicular Na(+)-dependent [3H]adenosine uptake was enhanced by an intravesicular-negative membrane potential and inhibited by dissipation of the Na+ gradient with gramicidin D. Both purine and pyrimidine nucleosides inhibited canalicular adenosine transport. 6-[(4-Nitrobenzyl)thio]-9-beta-D-ribofuranosylpurine, an inhibitor of nucleoside transport in erythrocytes and nonepithelial cells, had no effect on canalicular adenosine transport. Canalicular Na(+)-dependent [3H]adenosine uptake exhibited saturability with a Michaelis-Menten constant of 8.3 microM and a maximum transport rate of 7.6 pmol.5 s-1.mg protein-1. In contrast, [3H]adenosine uptake in blLPM vesicles was not stimulated by an inwardly directed Na+ gradient. These findings demonstrate asymmetric distribution of hepatic Na(+)-dependent nucleoside transport. Reclamation of intracanalicular adenosine resulting from ecto-ATPase activity may explain the presence of this transport process selectively on the bile canalicular membrane.