This study characterized the hepatobiliary disposition of 5 (and 6)-carboxy-2',7'-dichlorofluorescein (CDF), a model Abcc2/Mrp2 (canalicular) and Abcc3/Mrp3 (basolateral) substrate, in perfused livers from male C57BL/6 wild-type, Abcg2-/-, and Abcc2-/- mice. After single-pass liver perfusion with 1 muM CDF diacetate for 30 min and an additional 30-min perfusion with CDF-free buffer, cumulative biliary excretion of CDF in Abcg2-/- mice was significantly higher than in wild-type mice (65 +/- 6 and 47 +/- 15% of dose, respectively, p < 0.05), whereas CDF recovery in bile of Abcc2-/- mice was negligible. Cumulative recovery of CDF in perfusate was significantly higher in Abcc2-/- (90 +/- 8% of dose) relative to wild-type (35 +/- 11% of dose) mice. Compartmental pharmacokinetic analysis revealed that the rate constant for CDF biliary excretion was significantly increased in Abcg2-/- (0.061 +/- 0.005 min(-1)) compared with wild-type (0.039 +/- 0.011 min(-1)) mice. The rate constant governing the basolateral excretion of CDF was approximately 4-fold higher in Abcc2-/- (0.12 +/- 0.02 min(-1)) relative to wild-type (0.030 +/- 0.011 min(-1)) mice but was not altered in Abcg2-/- (0.031 +/- 0.004 min(-1)) mice. Hepatic Abcc3 protein levels, determined by immunoblot analysis, were approximately 60% higher in Abcc2-/- mice than in wild-type mice. In contrast, neither Abcc3 protein levels nor Abcc2 mRNA levels were altered in Abcg2-/- relative to wild-type mice. These data in knockout mouse models demonstrate that loss of expression and function of one canalicular transport protein may change the route and/or extent of excretion into bile or perfusate because of alterations in the function of other basolateral or canalicular transport proteins.