Influx kinetics and inhibitor specificity have been compared for the transport of methotrexate, 5-methyltetrahydrofolate, and folate in CCRF-CEM human lymphoblastoid cells. Influx of each folate compound proceeded with approximately the same V max, fluctuated in the same fashion with the ionic composition of the medium, and was blocked by low concentrations of an N-hydroxysuccinimide ester of methotrexate in both an anion-deficient buffer and in a buffered saline medium containing physiological concentrations of glucose and bicarbonate. Moreover, methotrexate influx was inhibited by 5-methyltetrahydrofolate and folate, and the inhibition constants ( K i ) of the latter compounds were equivalent to their K t values for half-maximal influx. Folate influx was likewise inhibited by methotrexate. The K i for methotrexate was equivalent to its K t for influx, and o-phthalate and phosphate each inhibited folate and methotrexate with the same degree of effectiveness. Various reversible and irreversible inhibitors reduced the influx of each folate substrate by >90%, and the progression of inhibition in each case was indicative of a single uptake component. Folate influx exhibited the same high sensitivity to inhibitors of methotrexate influx when measurements were performed at folate concentrations near the K t for influx (10–50 μM) or at concentrations approximating physiological conditions (5–20 nM). These results indicate that CCRF-CEM cells possess a single shared transport system for the uptake of methotrexate, 5-methyltetrahydrofolate, and folate and that other high- or low-affinity uptake processes are not present in these cells.