The fluorescent organic anion fluorescein (FL) accumulates in proximal tubular cells of the kidney during renal secretion. In freshly isolated and permeabilized proximal tubular cells, the uptake was reduced but still sensitive to probenecid, suggesting a concentrative mechanism that is associated with intracellular compartments. Previous studies have shown that one of these compartments may be mitochondrial. In this study, we further investigated the transport characteristics of FL in isolated rat kidney cortex mitochondria. Mitochondrial uptake of 100 microM FL was rapid, with an initial rate of 60 pmol/mg protein.min, and reached equilibrium after 5 min. To characterize the transport system(s) involved, FL uptake was studied in the absence and presence of substrates or inhibitors specific for the various mitochondrial anion carriers. Phenylsuccinate (10 mM), an inhibitor of the alpha-ketoglutarate carrier, reduced uptake significantly with a maximum inhibition of 33% and an inhibitory constant (-log IC(50)) of 4.0 +/- 0.4 (P <.05). The apparent K(m) for the phenylsuccinate-corrected FL uptake was 1.3 +/- 0.3 mM with a V(max) of 260 +/- 26 pmol/mg protein.15 s. Substrates for the tricarboxylate and glutamate-aspartate carriers significantly reduced the uptake of 100 microM FL with -log IC(50) values of 4.6 +/- 0.4 (citrate), 5.5 +/- 0.3 (glutamate), and 4.1 +/- 0.4 (aspartate). Substrates for the monocarboxylate and dicarboxylate carriers were without effect. The anionic drugs, valproate, indomethacin, and salicylate, significantly reduced FL uptake, whereas cephaloglycin and cephaloridine had no effect. Finally, a combination of phenylsuccinate, glutamate, and citrate reduced the uptake by 66%, indicating that at least three metabolite carriers contribute concomitantly to intramitochondrial FL transport.