ATP-binding cassette G2 protein (ABCG2, also known as breast cancer resistance protein) is expressed in various tissues including intestine, liver and kidney. Abcg2 has been shown to play a predominant role in the biliary excretion and pharmacokinetics of nitrofurantoin in mice (Merino et al, Mol Pharmacol 67:1758, 2005). In the present study, we evaluated the contribution of Abcg2 to the biliary excretion and pharmacokinetics of three Abcg2 substrates (nitrofurantoin and proprietary compounds A and B) in Abcg2 knockout (KO) rats. Methods: Biliary and urinary excretion of the three test compounds following IV administration was determined in bile duct catheterized Abcg2 KO and wild-type (WT) Sprague-Dawley rats (Sigma SageTM lab). In vitro bi-directional transport was performed in MDR1-LLC-PK1 and ABCG2-MDCK cells at 5 µM in the presence of 0.1% BSA. Results: Nitrofurantoin had efflux ratios of 1.7 and ~10 in MDR1-LLC-PK1 and ABCG2-MDCK cells, respectively. Compounds A and B were substrates for both MDR1 and ABCG2 (efflux ratio >10). Biliary excretion of nitrofurantoin accounted for 1.6% of the dose in the WT rats and was reduced 10-fold in the Abcg2 KO animals. In contrast, urinary excretion accounted for 18.2% of the dose in the WT rats and was similar in the Abcg2 KO animals. In the WT rats, approximately 40% and 50% of the dose was excreted into bile as parent for compounds A and B, respectively. Consistent with the corresponding in vitro efflux status, biliary excretion was decreased 3 to 5-fold in the Abcg2 KO rats. In the intact animals, clearance of nitrofurantoin, compound A and compound B was 26%, 32% and 58% lower in the Abcg2 KO rats as compared to that in the WT animals, respectively. Oral absorption of nitrofurantoin (10 mg/kg) was high and comparable in both WT and Abcg2 KO rats. Conclusion: Bcrp played an important role in the biliary, but not urinary, excretion of Abcg2 substrates nitrofurantoin compound A and compound B in rats. However, the impact of Bcrp on clearance was minimal to modest for these compounds. Although further studies are needed, initial results indicate that Abcg2 KO rats may be a useful model for understanding the role of Abcg2 in drug disposition.