Adenosine triphosphate (ATP) transporter from rat liver rough endoplasmic reticulum (RER) was solubilized and reconstituted into phosphatidylcholine liposomes. The RER proteoliposomes, resulting from optimizing some reconstitution parameters, had an apparent K m value of 1.5 μM and a V max of 286 pmol min −1 (mg protein) −1 and showed higher affinity for ATP and a lower V max value than intact RER ( K m of 6.5 μM and V max of 1 nmol). ATP transport was time- and temperature-dependent, inhibited by 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid, which is known as an inhibitor of anion transporters including ATP transporter, but was not affected by atractyloside, a specific inhibitor of mitochondrial ADP/ATP carrier. The internal and external effects of various nucleotides on the ATP transport were examined. ATP transport was cis-inhibited strongly by ADP and weakly by AMP. ADP-preloaded RER proteoliposomes showed a specific increase of ATP transport activity while AMP-preloaded RER proteoliposomes did not show the enhanced overshoot peak in the ATP uptake plot. These results demonstrate the ADP/ATP antiport mechanism of ATP transport in rat liver RER.