We have characterized the substrate specificity and mechanism of transport of the human multidrug resistance associated protein 3 (MRP3). A murine fibroblast-like cell line generated from the kidney of mice that lack Mdr1a/b and Mrp1 was retrovirally transduced with MRP3 cDNA. Stable clones overproducing MRP3 were resistant to the epipodophyllotoxins etoposide and teniposide, but not to vincristine, doxorubicin and cisplatin, drugs suggested to be MRP3 substrates by others. The resistance to etoposide was associated with reduced cellular accumulation and enhanced efflux of this drug, was not affected by depleting cells of glutathione but was inhibited by several common organic anion transport inhibitors. Membrane vesicles from infected insect cells expressing MRP3 mediated ATP-dependent transport of estradiol 17-b-D glucuronide, leukotriene C4, dinitrophenyl S-glutathione, but not glutathione itself, and of etoposide glucuronide, a major metabolite of etoposide in vivo. The transport of estradiol 17-b-D glucuronide by MRP3 was inhibited in a concentration dependent manner by both etoposide and methotrexate. Even though etoposide glucuronide is an excellent substrate for MRP3, this compound is not involved in the etoposide resistance of our MRP3 cells, as these cells extrude unmodified etoposide rather than etoposide glucuronide.