In order to identify the ATP transporter in rat liver rough endoplasmic reticulum (RER), a photoreactive azido derivative of ATP, 3‘-O-(p-azidobenzoyl)-ATP (AB-ATP), was synthesized by the reaction of ATP with N-hydroxysuccinimido 4-azidobenzoate (NHS-AB). The activity of the ATP transporter was determined by measuring the influx of [8-¹⁴C]ATP. The ATP transport had an apparent K_m value of 6.5 μM and a V_max of 1 nmol min^-1 (mg of protein)^-1. The transport of ATP was specifically inhibited by AB-ATP and 4,4‘-diisothiocyanatostilbene-2,2‘-disulfonic acid (DIDS). Under a dim light, AB-ATP was a competitive inhibitor of the ATP transport with a K_i value of 0.19 μM, which indicates that AB-ATP has a high affinity for the ATP transporter, so it can be utilized as a photoaffinity probe for the identification of the ATP transporter in rat liver RER. An SDS−PAGE analysis of RER vesicles photolabeled with [γ-³²P]AB-ATP indicates the presence of a 56-kDa protein. The 56-kDa protein was completely protected from photoaffinity labeling by 10 μM ATP but not by 30 μM GTP. The specific labeling of the 56-kDa protein was sensitive to the anion transport inhibitor DIDS. In order to confirm whether the apparent uptake of ATP was due to the 56-kDa protein, the ATP transporter was partially purified through two successive ion-exchange chromatography steps (DEAE and Mono-S). The fraction showing the high activity of the ATP transporter also contained the 56-kDa protein photolabeled with [γ-³²P]AB-ATP. On the basis of the photoaffinity labeling and reconstitution experiment, we conclude that the 56-kDa protein represents the ATP transporter in rat liver RER.