The bioavailability and targeted distribution of abacavir (ABC) and zidovudine (AZT) to viral reservoirs may be influenced by efflux transporters. The purpose of this study was to characterize the interaction of these nucleoside reverse transcriptase inhibitors with the Abcg2/Bcrp1 transporter, the murine homolog of human breast cancer resistance protein (BCRP), using a Bcrp1-transfected Madin-Darby canine kidney II cell model. Intracellular accumulation of ABC and AZT was significantly reduced by ∼90% and ∼70%, respectively, in Bcrp1-transfected cells compared with the wild-type cells. Both ABC and AZT showed significantly increased basolateral-to-apical (B-to-A) and decreased apical-to-basolateral (A-to-B) transport in Bcrp1 cells compared with wild-type directional flux. The efflux ratio (ratio of B-to-A to A-to-B) in Bcrp1-transfected cells was 22 for ABC and 11 for AZT. N-(4-[2-(1,2,3,4- tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy- 9-oxo-4-acridine carboxamide (GF120918) inhibited this difference in accumulation between the two cell variants with an EC50 of 1.32 ± 0.3 μM for ABC and 0.31 ± 0.1 μM for AZT. Potent and highly cooperative inhibition by Ko143 (3-(6-isobutyl-9-methoxy-1,4-dioxo-1,2,3, 4,6,7,12,12a-octahydropyrazino[1′,2′:1,6]pyrido[3,4-b]indol-3-yl) -propionic acid tert-butyl ester) was observed with an EC50 of 121 ± 5 nM for ABC and 19.2 ± 1.5 nM for AZT (Hill coefficient ∼3-6). Probenecid, an organic anion inhibitor known to influence AZT biodistribution, had no effect on cellular accumulation in the Bcrp1 model. These studies characterize the Bcrp1-mediated transport of ABC and AZT and show that prototypical BCRP inhibitors GF120918 and Ko143 can inhibit the Bcrp1-mediated transport of these important antiretroviral compounds. The functional expression of BCRP at critical barriers, such as the intestinal enterocytes, brain capillary endothelium, and target lymphocytes, could influence the bioavailability and targeted delivery of these drugs to sanctuary sites.