Gefitinib is an orally active inhibitor of the epidermal growth factor receptor approved for use in patients with locally advanced or metastatic non-small cell lung cancer. It has also been evaluated in several clinical trials for treatment of brain tumors such as high-grade glioma. In this study, we investigated the influence of P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) on distribution of gefitinib to the central nervous system. In vitro studies conducted in Madin-Darby canine kidney II cells indicate that both P-gp and BCRP effectively transport gefitinib, limiting its intracellular accumulation. In vivo studies demonstrated that transport of gefitinib across the blood-brain barrier (BBB) is significantly limited. Steady-state brain-to-plasma (B/P) concentration ratios were 70-fold higher in the Mdr1a/b(-/-) Bcrp1(-/-) mice (ratio of approximately 7) compared with wild-type mice (ratio of approximately 0.1). The B/P ratio after oral administration increased significantly when gefitinib was coadministered with the dual P-gp and BCRP inhibitor elacridar. We investigated the integrity of tight junctions in the Mdr1a/b(-/-) Bcrp1(-/-) mice and found no difference in the brain inulin and sucrose space between the wild-type and Mdr1a/b(-/-) Bcrp1(-/-) mice. This suggested that the dramatic enhancement in the brain distribution of gefitinib is not due to a leakier BBB in these mice. These results show that brain distribution of gefitinib is restricted due to active efflux by P-gp and BCRP. This finding is of clinical significance for therapy in brain tumors such as glioma, where concurrent administration of a dual inhibitor such as elacridar can increase delivery and thus enhance efficacy of gefitinib.