ABCG2 is an ATP-binding cassette transporter that confers resistance to various chemotherapeutic agents. Recent studies have established that an Arg (wild-type) to Gly mutation at amino acid 482 in ABCG2 alters substrate specificity. Here, we explored the role of this G482 mutation in antifolate resistance using a clinically relevant 4-hour drug exposure. Stable transfectants overexpressing the mutant G482 transporter displayed 120-, 1,000-, and >6,250-fold resistance to the antifolates methotrexate, GW1843, and Tomudex, respectively, relative to parental human embryonic kidney cells. Moreover, although overexpressing equal transporter levels at the plasma membrane, G482-ABCG2 cells were 6-, 23-, and >521-fold more resistant to methotrexate, GW1843, and Tomudex, respectively, than R482-ABCG2 cells. In contrast, upon a continuous (72-hour) drug exposure, both the G482- and R482-ABCG2 cells lost almost all their antifolate resistance; this result was consistent with the inability of ABCG2 to extrude long-chain antifolate polyglutamates. Ko143, a specific and potent ABCG2 inhibitor reversed methotrexate resistance in both G482- and R482-ABCG2 cells. Consistently, whereas the pool of free methotrexate in parental human embryonic kidney cells was prominent after 4 hours of transport with 1 micromol/L [3H]methotrexate, in R482- and G482-ABCG2 cells, it was minimal. Furthermore, G482-ABCG2 cells contained marked decreases in the di- and triglutamate species of [3H]methotrexate at 4 hours of incubation with methotrexate and in the tetra- and pentaglutamates at 24 hours. These changes were not associated with any significant decrease in folylypoly-gamma-glutamate synthetase activity. These results provide the first evidence that the G482-ABCG2 mutation confers high-level resistance to various hydrophilic antifolates.