Human ATP synthase (complex V) is a multi-subunit enzyme complex that consists of a catalytic portion (F1), a membrane portion (F0) and two stalks linking F1 and F0. Complex V uses the electrochemical potential created by the oxidative phosphorylation (OXPHOS) complexes I–IV to synthesize ATP. Following blue native polyacrylamide gel electrophoresis (BN-PAGE), the activities of OXPHOS complexes can be evaluated. In part of the patients with suspected OXPHOS defects, catalytically active subcomplexes of complex V are detected. Complex V is peculiarly fragile to defects in mitochondrial protein synthesis, as its two mtDNA encoded structural subunits are anchoring proteins linking the catalytic domain to the mitochondrial inner membrane. We speculated that complex V subcomplexes are selective indicators of defects in mtDNA, affecting intra-mitochondrial protein synthesis. To test this hypothesis, we evaluated in retrospect the BN-PAGE activity patterns in tissue samples from the patients with either deficient OXPHOS complex activities documented by spectrophotometric analysis, or from the patients suspected on a clinical basis of having a mitochondrial defect. The BN gels were incubated with specific staining solutions to evaluate OXPHOS complex activities, and to detect catalytically active complex V subcomplexes. Patients with complex V subcomplexes were screened for abnormalities in the mtDNA. More then 460 tissue samples originating from 440 patients (227 muscle samples, 186 fibroblast strains and 51 heart and liver samples) were investigated. In 51 tissues originating from 42 patients, complex V subcomplexes were detected. In 22 of these patients, a defect in mtDNA was detected: tRNA mutations (10), mtDNA deletion (1), ATP6 mutation (4), and mtDNA depletion (7). BN-PAGE combined with in-gel activity staining is a convenient method to distinguish OXPHOS defects originating from mtDNA mutations. The presence of complex V subcomplexes represents a diagnostic criterium for defective intra-mitochondrial protein synthesis.