In the present work we used 8-azido [alpha-P-32] ATP to identify the uncoupling protein of potato tuber mitochondria (PUMP). An adenine nucleotide binding site is described which is distinct from the one present in the ATP/ADP carrier (AAC). This adenine nucleotide binding site is sensitive to GTP with a dissociation constant for 8-azido [alpha-P-32] ATP of 21 mu M and an optimum at acidic pH. The GTP sensitivity and pH dependence of the adenine nucleotide binding site detected in potato tuber mitochondria was not apparent in rat liver mitochondria (RLM), and closely corresponds to the uncoupling mitochondrial protein (UCP) of brown adipose tissue mitochondria (BATM). The probe 8-azido [alpha-P-32] ATP was covalently incorporated into potato tuber mitochondria by near ultraviolet irradiation. Two major radioactive bands of 30,000 and 32,000 Da were identified in the autoradiography of the SDS gel electrophoresis of potato mitochondrial proteins. These bands correspond to AAC and PUMP, since labelling was prevented by carboxyatractyloside (CAT) or GTP, respectively.359701706