The uptake system for 6-diazo-5-oxo-L-norleucine (DON) was studied in mouse P388 leukemia cells. The DON transport system was found to resemble that of another glutamine antimetabolite, Acivicin, in its strong temperature dependence, utilization of the “L” transport system, inhibition by glutamine but not by glutamate, potent inhibition by p-chlo-romercuribenzene sulfonate, Na+, and only minimal inhibition by various energy poisons. A Km of approximately 70 μm and a Vmax0, of 3.4 nmoles/106 cells/min was calculated for this cell line. The accumulated DON was not metabolized by P388 cells and moderate efflux occurred at 37°C. The DON transport characteristics of a DON-resistant P388 cell line (100 times ID50 of parent line) were similar to those of the DON-sensitive parent line, indicating that altered drug transport may not be involved in development of resistance to this antimetabolite. The finding that an Acivicin-resistant subline of P388 cells which exhibited good transport of DON showed negligible transport of Acivicin suggests different modes of resistance towards the two glutamine and metabolites.