Tamoxifen is widely prescribed to patients with estrogen receptor-positive breast cancer, and it is a prodrug that requires bioactivation by cytochrome P450 enzymes CYP2D6 and 3A4 to generate the active metabolite, endoxifen. Large interpatient variability in endoxifen plasma levels has been reported, and polymorphisms in CYP2D6 have been implicated as a major determinant of such variability. However, little is known regarding the role of drug transporters such as P-glycoprotein [multidrug resistance 1 (MDR1), ATP-binding cassette B1 (ABCB1)] to endoxifen disposition and response. Therefore, we determined the ability of P-glycoprotein to transport endoxifen in vitro, using a polarized human P-glycoprotein-overexpressing cell line. Markedly higher transport of endoxifen was observed in the basal-to-apical direction, which was abrogated in the presence of the potent and specific P-glycoprotein inhibitor (2R)-anti-5-{3-[4-(10,11-difluoromethanodibenzo-suber-5-yl)piperazin-1-yl]-2-hydroxypropoxy}quinoline trihydrochloride (LY335979). To validate the in vivo relevance of P-glycoprotein to endoxifen disposition, plasma and tissue concentrations were also determined in Mdr1a-deficient mice after oral administration of endoxifen. Plasma endoxifen levels did not significantly differ between wild-type and Mdr1a-deficient mice. However, brain concentrations of endoxifen were nearly 20-fold higher in Mdr1a-deficient mice compared to wild-type mice. Because P-glycoprotein is highly expressed at the blood-brain barrier and in some breast cancer tumors, variation in expression and function of this transporter may alter central nervous system entry and the attained intracellular concentration in such breast cancer cells and therefore may prove to be of relevance to therapeutic outcome.