Previous studies implicated P-glycoprotein (P-gp) as the major transport protein responsible for the biliary excretion of fexofenadine (FEX). However, FEX biliary excretion was not impaired in P-gp- or breast cancer resistance protein (Bcrp)-knockout mice or multidrug resistance-associated protein 2 (Mrp2)-deficient rats. The present study tested the hypothesis that species differences exist in the transport protein primarily responsible for FEX biliary excretion between mice and rats. Livers from Mrp2-knockout (Mrp2KO) mice and Mrp2-deficient (TR(-)) rats were perfused in a single-pass manner with 0.5 muM FEX. N-(4-[2-(1,2,3,4-Tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918) (10 muM) was employed to inhibit P-gp and Bcrp. The biliary excretion rate of FEX was decreased 85% in Mrp2KO relative to wild-type mice (18.4 +/- 2.2 versus 122 +/- 34 pmol/min/g liver). In mice, more than 50% of FEX unbound intrinsic biliary clearance (CL(bile, int)(') = 3.0 ml/h/g liver) could be attributed to Mrp2 (Mrp2-dependent CL(bile, int)(') approximately 1.7 ml/h/g liver), with P-gp and Bcrp playing a minor role (P-gp- and Bcrp-dependent CL(bile, int)(') approximately 0.3 ml/h/g liver). Approximately one third of FEX CL(bile, int)(') was attributed to unidentified mechanisms in mice. In contrast to mice, FEX biliary excretion rate (245 +/- 38 and 250 +/- 25 pmol/min/g liver) and CL(bile, int)(') (9.72 +/- 2.47 and 6.49 +/- 0.68 ml/h/g liver) were comparable between TR(-) and control Wistar rats, respectively, suggesting that unidentified transport mechanism(s) can completely compensate for the loss of Mrp2 function in rats. Mrp2 clearly plays a major role in FEX biliary excretion in mice. In conclusion, remarkable species differences exist in FEX hepatobiliary transport mechanisms.