To characterize the transport functions of a recently cloned basolateral organic anion transporting polypeptide of rat hepatocytes we performed further kinetic transport and substrate cis-inhibition studies in organic anion-transporting polypeptide-cRNA injected Xenopus Zaevis oocytes. The studies demonstrate saturable N a + -independent sulfobromophthalein (Michaelis-Menten constant, 1.5 pmolL) and taurocholate (Michaelis-Menten constant, 50 Fmol/L) uptake by organic anion-transporting polypeptide. Sulfobromophthalein uptake was inhibited by the following organic anions: 0.01 mmolL bilirubin (43%), 0.1 mmol/L indocyanine green (S1%), 0.1 mmoln 4,4'diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS; 52%) and 1 mmol/L probenecid (74%). Competitive inhibition was shown for indocyanine green (inhibition constant about 1.3 pmol/L). Sulfobromophthalein and taurocholate uptakes were also inhibited by cholate, chenodeoxycholate, deoxycholate and ursodeoxycholate, as well as their glycine and taurine conjugates. Organic anion-transporting polypeptide also mediated uptake of glycocholate, tauroursodeoxycholate and taurochenodeoxycholate. No cis-inhibition of sulfobromophthalein uptake was seen in the presence of ATP, para-aminohippuric acid, bumetanide, digitoxin, reduced glutathione, leukotriene C,, nicotinic acid, ouabain, oxalate, rifampicin, succinate or sulfate. Furthermore, radioactively labeled paraaminohippuric acid, a-ketoglutarate and reduced glutathione were not taken up by organic anion-