Abstract Purpose Multidrug resistance (MDR) impedes cancer treatment. Two efflux transporters from the ATP-binding cassette (ABC) family, ABCB1 and ABCG2, may contribute to MDR by restricting the entry of therapeutic drugs into tumor cells. Although a higher expression of these transporters has been correlated with an unfavorable response to chemotherapy, transporter expression does not necessarily correlate with function. In this study, we characterized the pharmacological properties of [¹⁸F]AVT-011, a new PET radiotracer for imaging transporter-mediated MDR in tumors. Methods AVT-011 was radiolabeled with ¹⁸F and evaluated with PET imaging in preclinical models. Transport of [¹⁸F]AVT-011 by ABCB1 and/or ABCG2 was assessed by measuring its uptake in the brains of wild-type, Abcb1a/b^−/−, and Abcg2^−/− mice at baseline and after administration of the ABCB1 inhibitor tariquidar (n = 5/group). Metabolism and biodistribution of [¹⁸F]AVT-011 were also measured. To measure ABCB1 function in tumors, we performed PET experiments using both [¹⁸F]AVT-011 and [¹⁸F]FDG in mice bearing orthotopic breast tumors (n = 7–10/group) expressing clinically relevant levels of ABCB1. Results At baseline, brain uptake was highest in Abcb1a/b^−/− mice. After tariquidar administration, brain uptake increased 3-fold and 8-fold in wild-type and Abcg2^−/− mice, respectively, but did not increase further in Abcb1a/b^−/− mice. At 30 min after injection, the radiotracer was > 90% in its parent form and had highest uptake in organs of the hepatobiliary system. Compared with that in drug-sensitive tumors, uptake of [¹⁸F]AVT-011 was 32% lower in doxorubicin-resistant tumors with highest ABCB1 expression and increased by 40% with tariquidar administration. Tumor uptake of [¹⁸F]FDG did not significantly differ among groups. Conclusion [¹⁸F]AVT-011 is a dual ABCB1/ABCG2 substrate radiotracer that can quantify transporter function at the blood-brain barrier and in ABCB1-expressing tumors, making it potentially suitable for clinical imaging of ABCB1-mediated MDR in tumors.