In isolated rabbit cortical renal tubules, phosphate transport rates change in response to the metabolic demands for inorganic phosphate. The mechanisms involved in this association may be related to changes in cytosolic concentration of inorganic phosphate. The present studies were designed to manipulate mitochondrial metabolism and to measure the rate of phosphate transport under these conditions. The measured change in rates of oxidative phosphorylation and dephosphorylation reactions was used to calculate the effects on the cytosolic concentration of inorganic phosphate. First, malate (1 mmol/L) was added to suspensions of rabbit cortical renal tubules to stimulate oxidative phosphorylation. This addition was associated with an increase in both oxidative phosphorylation and ouabain-sensitive dephosphorylation, but the effect on phosphorylation was greater and the calculated demand for cytosolic phosphate was increased. Under these conditions, isolated, perfused proximal convoluted tubules absorbed phosphate at a faster rate (6.9 +/- 1.6 v 4.9 +/- 1.0 pmol/mm/min; P less than 0.05). Second, n-butylmalonate (5 mmol/L) was added to suspensions of tubules to partially inhibit mitochondrial transport of anions and oxidative phosphorylation. This manipulation was associated with an equivalent decrease in both phosphorylation and dephosphorylation rates and no change in the demand for cytosolic phosphate. In perfused proximal convoluted tubules, fluid absorption was reduced but the phosphate transport rate was unchanged (5.3 +/- 1.2 v 4.4 +/- 0.9 pmol/mm/min). These data are consistent with the hypothesis that alterations in the cytosolic demand for inorganic phosphate participate in the regulation of the phosphate transport mechanism of proximal convoluted tubules.