These studies examine the phosphate dependence of fluid absorption and oxidative metabolism in proximal tubules from rabbit kidney. Removal of inorganic phosphate from intraluminal fluid in isolated perfused tubules reduced fluid absorption (Jv) from 1.0 nl X mm-1 X min-1 to zero and removal of phosphate from the medium bathing tubules in suspension reduced O2 consumption rates (coupled, uncoupled, ouabain sensitive, and ouabain insensitive) and ATP contents by 30-40%. Inhibitions induced by removal of phosphate could be altered by addition of selected organic substrates to ambient fluids. Addition of succinate, citrate, or malate preserved uncoupled respiration and partially preserved Jv (0.4-0.9 nl X mm-1 X min-1), with succinate being most effective. Addition of short-chain fatty acids (butyrate or valerate) to phosphate-free media had little or no effect on Jv. n-Butylmalonate, an inhibitor of dicarboxylate anion transport into mitochondria, reduced oxidative metabolism, ATP content, and Jv when added to phosphate-containing media but had no further effect on these tubular functions when added to phosphate-free media. Together these results suggest that phosphate limitation reduces dicarboxylate anion availability to mitochondria and that this inhibition may contribute to the overall reduction in oxidative metabolism and Jv observed under these conditions.