Increasing concentrations of 2,4-dinitrophenol exert a biphasic effect on the O2 consumption of mitochondria oxidizing certain carboxylic acid substrates in 0.15 m KCl in the presence of adenosine triphosphate (ATP) or adenosine diphosphate (ADP) and Mg2+. This effect of 2,4-dinitrophenol is modified by a soluble cytoplasmic cellular fraction. The modification consists of a large increase of O2 consumption within a limiting range of concentrations of 2,4- dinitrophenol. The cytoplasmic component responsible for this effect was obtained from rat liver, kidney, and heart by gel filtration on Sephadex G-25 and G-50 as a macromolecular complex of apparent molecular weight of 5000-6000. The metabolically active cytoplasmic fraction inhibits the activating effect of 2,4- dinitrophenol on mitochondrial adenosine triphosphatase (ATPase) only in the presence of carboxylic acid substrates. Prerequisities for in vitro metabolic action of the cytoplasmic fraction are preservation of mitochondrial membrane system and presence of ADP or ATP (adenosine monophosphate is ineffective), isotonic KCl (present both during preparation of the fraction and during metabolic experiments), and a specific concentration of 2,4-dinitrophenol, which is characteristic for certain substrates and varies with the tissue source of mitochondria. A possible interpretation of these results is proposed in terms of extramitochondrial regulation of mitochondrial permease systems.