The flux control coefficients of adenine nucleotide translocase, the phosphate transporter, and H(+)-ATPase were determined in rat skeletal muscle mitochondria using glutamate plus malate as substrates and soluble F1-ATPase as load enzyme. It was observed that the flux control coefficients of adenine nucleotide translocase, H(+)-ATPase, and the load enzyme F1-ATPase, at comparable rates of respiration, strongly depend on the phosphate concentration in the incubation medium. So, the flux control exerted by adenine nucleotide translocase, in the intermediate states of mitochondrial respiration (approximately 120 nmol of O2/min/mg) at 10 mM phosphate, was found to be about 0.37. At a phosphate concentration of 1 mM and comparable rates of respiration the flux control coefficient of the translocase decreased to about 0.20. Under these conditions, a sharp increase in the controlling influence of H(+)-ATPase from 0.10 to 0.74 was detected. Furthermore, at this flux rate, the sum of flux control coefficients of adenine nucleotide translocase, H(+)-ATPase, phosphate transporter, and the load enzyme F1-ATPase was noted to be very close to unity. This indicates that under the conditions of intermediate state respiration, all of the other reactions have a negligible controlling influence on oxidative phosphorylation in skeletal muscle mitochondria.