This review highlights current insights into the regulation of the mitochondrial respiratory chain (electron transport chain, ETC) activity. The regulation of ETC properties optimizes ATP synthesis and controls the generation of the superoxide anion radical (O ) which can be converted into other reactive oxygen species (ROS) playing a dual role by initiating signaling cascades or contributing to oxidative stress. We examine how ETC activity is influenced by the structure and conformation of its complexes, their allosteric or post-translational modifications, and their interactions with membrane lipids. The formation and function of supercomplexes, as well as their cell-type-specific characteristics, are also discussed, alongside with the role of intracellular Ca concentration in the modulation of ETC activity. Furthermore, we discuss mechanisms and sites of O generation within ETC complexes, O fate in the mitochondrial matrix, and the impact of cytochrome c (Cyt c) conformation and allosteric modifications on ETC function. Finally, we discuss various abnormalities in ETC complexes, emphasizing their relevance to mitochondrial dysfunction and disease.