Updated project metadata. The mammalian oxidative phosphorylation (OXPHOS) system in the inner mitochondrial membrane comprises respiratory chain complexes I-IV (CI-CIV) and ATP synthase. Together, these protein complexes harvest metabolic energy to generate ATP, the cellular energy currency. The inner mitochondrial membrane is abundant in OXPHOS complexes and CI, CIII and CIV exhibit specific protein-protein interactions to form stable supercomplex assemblies, exemplified by the respirasome (CI-CIII2-CIV). Respirasomes are conserved in evolution, and as well documented by biochemical and structural methods. However, their physiological roles are much debated, despite a substantial literature suggesting their importance in facilitating catalysis and regulating turnover in response to metabolic demand. To investigate the in vivo role of respirasomes, we deleted a short conserved, charged loop in the UQCRC1 subunit of CIII that contacts CI. The resulting homozygous knock-in mice show profoundly decreased levels of respirasomes on blue native polyacrylamide gel electrophoresis (BN-PAGE) and complexome profiling proteomics analyses of different tissues, although the individual complexes appear unaffected. In vivo The spatial organization of respirasomes in vivo was altered in knock-in mice as shown by cross-linking experiments on intact mitochondria. Surprisingly, the mutant mice are healthy, with normal respiratory chain capacity and normal exercise tolerance. Therefore, respirasomes are dispensable for OXPHOS function in vivo.