Abstract Purpose: The brain is a pharmacologic sanctuary site, due to the presence of the blood-brain barrier (BBB). Whereas the effect of P-glycoprotein (P-gp) at the BBB is well established, the role of breast cancer resistance protein (BCRP) that is also expressed at the BBB is not. Experimental Design: We have studied the effect of BCRP by administering topotecan to wild-type (WT), single Mdr1a/b(−/−) and Bcrp1(−/−), and compound Mdr1a/b(−/−)Bcrp1(−/−) knockout mice. Drug levels in plasma and tissues were determined by high-performance liquid chromatography. Results: The area under the plasma and tissue concentration-time curve (AUC) of topotecan in brains of Mdr1a/b(−/−) and Bcrp1(−/−) mice was only 1.5-fold higher compared with WT mice, but in Mdr1a/b(−/−)Bcrp1(−/−) mice, where both transporters are absent, the AUC increased by 12-fold. The AUC in plasma was ∼0.75-, 2.4-, and 3.7-fold higher in Mdr1a/b(−/−), Bcrp1(−/−), and Mdr1a/b(−/−)Bcrp1(−/−) mice, respectively, resulting in 2.0-fold (P < 0.01), 0.65-fold (P, not significant), and 3.2-fold (P < 0.01), respectively, higher brain-to-plasma AUC ratios. Results using Mrp4(−/−) mice showed that this transporter had no effect on the brain penetration of topotecan. The P-gp/BCRP inhibitor elacridar fully inhibited P-gp–mediated transport of topotecan, whereas inhibition of Bcrp1-mediated transport by elacridar was minimal. Conclusions: Our results using Mdr1a/b(−/−)Bcrp1(−/−) mice clearly show the effect of Bcrp1 at the BBB and also show how two drug transporters act in concert to limit the brain penetration of topotecan. We expect that this finding will also apply to other drugs that are substrates of both P-gp and BCRP. Consequently, to improve the brain penetration of such compounds for targeting intracranial malignancies in patients, it will be essential to use potent inhibitors of both drug transporters.