Multidrug resistance (MDR1) P-glycoprotein (Pgp), multidrug resistance-associated protein (MRP1), and breast cancer resistance protein (BCRP/MXR/ABCP) are members of the ATP-binding-cassette (ABC) superfamily of membrane transporters and are thought to function as energy-dependent efflux pumps of a variety of structurally diverse chemotherapeutic agents. We herein report the characterization of ^99mTc-Tetrofosmin, a candidate radiopharmaceutical substrate of ABC transporters. ^99mTc-Tetrofosmin showed high membrane potential-dependent accumulation in drug-sensitive KB 3–1 cells and low antagonist-reversible accumulation in MDR KB 8–5 and KB 8–5-11 cells in proportion to levels of MDR1 Pgp expression. In KB 8–5 cells, ec₅₀ values of the potent MDR antagonists 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), (2R)-anti-5-{3-[4-(10,11-difluoromethanodibenzo-suber-5-yl)piperazin-1-yl]-2-hydroxypropoxy}quinoline trihydrochloride (LY335979), and (3′-keto-Bmt′)-[Val²]-cyclosporin A (PSC 833) were 40, 66, and 986 nM, respectively. Furthermore, only baculoviruses carrying human MDR1, but not MDR3, conferred both a decrease in accumulation of ^99mTc-Tetrofosmin in host Spodoptera frugiperda (Sf9) cells and a GF120918-induced enhancement. Transport studies with a variety of stably transfected and drug-selected tumor cell lines were performed with ^99mTc-Tetrofosmin and compared with ^99mTc-Sestamibi, a previously validated MDR imaging agent. MDR1 Pgp readily transported each agent. To a lesser extent, MRP1 also transported each agent, likely as co-transport substrates with GSH; neither agent was a substrate for the BCRP/MXR/ABCP half-transporter. In mdr1a(−/−) and mdr1a/1b(−/−) mice, ^99mTc-Tetrofosmin showed ∼3.5-fold greater brain uptake and retention compared with wild-type, with no net change in blood pharmacokinetics, consistent with transport in vivo by Pgp expressed at the capillary blood–brain barrier. Molecular imaging of the functional transport activity of ABC transporters in vivo with ^99mTc-Tetrofosmin and related radiopharmaceuticals may enable non-invasive monitoring of chemotherapeutic and MDR gene therapy protocols.