A cDNA encoding the new member of the multispecific organic anion transporter family, OAT3, was isolated by the reverse transcription-polymerase chain reaction cloning method. Degenerate primers were designed based on the sequences conserved among OAT1, OAT2, and organic cation transporter 1 (OCT1), and reverse transcription-polymerase chain reaction was performed using rat brain poly(A)+ RNA. The 536-amino acid protein sequence encoded by OAT3 showed 49, 39, and 36% identity to those of OAT1, OAT2, and OCT1, respectively. Northern blot analysis revealed that rat OAT3 mRNA is expressed in the liver, brain, kidney, and eye. When expressed in Xenopus laevis oocytes, OAT3 mediated the uptake of organic anions, such as p-aminohippurate (Km = 65 microM), ochratoxin A (Km = 0.74 microM), and estrone sulfate (Km = 2.3 microM) and a cationic compound, cimetidine. OAT3-mediated uptake of [3H]estrone sulfate was sodium-independent. para-Aminohippuric acid, estrone sulfate or ochratoxin A did not show any trans-stimulatory effect on either influx or efflux of [3H]estrone sulfate via OAT3. Organic anions such as sulfobromophthalein, probenecid, indocyanine green, bumetanide, piroxicam, furosemide, azidodeoxythymidine, 4, 4'-diisothiocyanostilbene-3,3'-disulfonic acid, and benzylpenicillin inhibited OAT3-mediated estrone sulfate uptake, while ouabain and digoxin did not. Organic cations such as tetraethylammonium, guanidine, verapamil, and quinidine did not interact with OAT3. Acidic metabolites of neurotransmitters derived from dopamine, epinephrine, norepinephrine, and serotonin inhibited the uptake of estrone sulfate via OAT3. These results suggest an important role of OAT3 in the excretion/detoxification of endogenous and exogenous organic anions, especially from the brain.