Liver mitochondria provided with an oxidizable substrate, ATP, oxygen, and an ADP-generating system (soluble F1-ATPase) were used to reevaluate the rate-controlling step(s) intrinsic to all of the processes of mitochondrial oxidative phosphorylation. The quantity termed "control strength" (C), previously defined as the fractional change in flux through a (system) induced by a fractional change in the concentration of an individual enzyme in the system, has been used to evaluate rate-influencing steps in this overall process by carefully defining the dimensions of the "system" under analysis. If the system is defined by a suspension of mitochondria provided with substrates, plus an extrinsic ADP-generating process (ATPase), the value of C of the latter for the overall process of phosphorylation-linked respiration is near 1.0 until the capacity of the mitochondria to phosphorylate ADP is approached, after which C for the soluble ATPase becomes zero as the maximum capacity for phosphorylation is attained. Carboxyatractyloside was found only marginally to inhibit respiration stimulated by ATPase, even when a large percentage of adenine nucleotide translocase molecules were immobilized. The relative lack of effect of carboxyatractyloside on phosphorylating respiration is explained by the readjustment of the concentration of one of the substrates (ADP) and an inhibitor (ATP), which results from inhibition of adenine nucleotide translocase. The residual blunted inhibition of respiration is explained by product inhibition of the ADP-regenerating ATPase, and not necessarily to any intrinsically mitochondrial intermediate process. The system being evaluated can be redefined to include only the processes intrinsic to mitochondria. This can be achieved by providing exactly comparable substrate concentrations to the mitochondria under comparable incubation conditions. Under these conditions, the adenine nucleotide translocase is the principal, if not the only, rate-controlling step in the overall process of oxidative phosphorylation until a new rate-limitation is attained (ATP synthesis). These data are consistent with the conclusion that, at intermediate rates of phosphorylation-coupled respiration, the extramitochondrial ATP/ADP ratio regulates this process through its kinetic effects on the catalytic properties of the adenine nucleotide translocase.