Recently, by analysis of mildly solubilized mitochondrial membranes new biochemical evidences were obtained for the occurrence of ATP synthase dimers in mitochondria of different eukaryotes from yeast to mammals. In the case of yeast even higher ATP synthase oligomers could be found. Here, we analysed by BN- and CN-PAGE mammalian (bovine and rat) mitochondria from five different tissues, which were efficiently but very mildly solubilized with digitonin. In mitochondria from all investigated tissues besides ATP synthase monomers (V-1) not only dimeric ATP synthase (V-2) but for the first time also higher oligomers, at least trimers (V-3) and tetramers (V-4), were separated. Compared with BN-PAGE, by CN-PAGE analysis the yields of preserved respiratory supercomplexes as well as of oligomeric ATP synthases (V2-4) were significantly increased. The latter represent the majority of total ATP synthases in all cases. Importantly, all different ATP synthase species from the five tissues displayed in-gel ATP hydrolase activity, suggesting that homooligomeric ATP synthases are the constitutive, enzymatically competent organization of mammalian ATP synthases in the inner mitochondrial membrane. (c) 2005 Elsevier Inc. All rights reserved.