Kidney proximal tubules develop a severe, but highly reversible energetic deficit due to nonesterified fatty acid (NEFA)-induced dissipation of mitochondrial membrane potential (DeltaPsi(m)) during reoxygenation after severe hypoxia. To assess the mechanism for this behavior, we have compared the efficacies of different NEFA for inducing mitochondrial de-energization in permeabilized tubules measured using safranin O uptake and studied modification of NEFA-induced de-energization by inhibitors of the ADP/ATP carrier and glutamate using both normoxic tubules treated with exogenous NEFA and tubules de-energized during hypoxia/reoxygenation. Among the long chain NEFA that accumulate during hypoxia/reoxygenation of isolated tubules and ischemia/reperfusion of the kidney in vivo, oleate, linoleate, and arachidonate had strong effects to dissipate DeltaPsi(m) that were slightly greater than palmitate, while stearate was inactive. This behavior correlates well with the protonophoric effects of each NEFA. Inhibition of the ADP/ATP carrier with either carboxyatractyloside or bongkrekic acid or addition of glutamate to compete for the aspartate/glutamate carrier improved DeltaPsi(m) in the presence of exogenous oleate and after H/R. Effects on the two carriers were additive and restored safranin O uptake to 80% of normal under both conditions. The data strongly support NEFA cycling across the inner mitochondrial membrane using anion carriers as the main mechanism for NEFA-induced de-energization in this system and provide the first evidence for a contribution of this process to pathophysiological events that impact importantly on energetics of intact cells.