To characterize and localize hepatic plasma membrane ATP-dependent Ca2+ transport and Na+/Ca2+ exchange, studies were performed using highly purified rat basolateral and canalicular membrane vesicles. ATP-dependent Ca2+ transport activity was present in vesicles from both domains, insensitive to azide, oligomycin, oxalate, calmodulin, and calmidazolium, and virtually abolished at pH 6.8. However, basolateral and canalicular transport differed significantly. While basolateral transport was markedly stimulated by 1 mM Mg2+, canalicular transport was Mg2+ independent. Basolateral transport was similar at pH 7.4 and 8.0 but canalicular activity was stimulated fourfold at pH 8.0. Both Ca2+ Km [1.4 +/- 0.1 (SE).10(-8) vs. 4.8 +/- 0.7.10(-8) M] and Vmax (3.6 +/- 0.1 vs. 9.0 +/- 0.6 nmol mg-1 protein min-1) were lower in basolateral than in canalicular vesicles. Basolateral transport was somewhat more nucleotide specific (for ATP) and sensitive to vanadate (IC50 130 vs. 500 microM, respectively) than was canalicular transport. Na+/Ca2+ exchange activity was not detected in membranes from either domain. These studies suggest that hepatic ATP-dependent Ca2+ transport is mediated by domain-specific carriers on the basolateral and canalicular membranes.