Multidrug resistance (MDR) seriously limits the efficacy of chemotherapy in patients with cancer and leukemia. Active transport across membranes is essential for such cellular drug resistance, largely provided by ATP-binding cassette (ABC) transport proteins. Intracellular drug sequestration contributes to MDR; however, a genuine intracellular ABC transport protein with MDR function has not yet been identified. Analyzing the intrinsic drug efflux capacity of leukemic stem cells, we found the ABC transporter A3 (ABCA3) to be expressed consistently in acute myeloid leukemia (AML) samples. Greater expression of ABCA3 is associated with unfavorable treatment outcome, and in vitro, elevated expression induces resistance toward a broad spectrum of cytostatic agents. ABCA3 remains localized within the limiting membranes of lysosomes and multivesicular bodies, in which cytostatics are efficiently sequestered. In addition to AML, we also detected ABCA3 in a panel of lymphohematopoietic tissues and transformed cell lines. In conclusion, we identified subcellular drug sequestration mediated by the genuinely intracellular ABCA3 as being a clinically relevant mechanism of intrinsic MDR.