F olate transport in phenylhydrazine-induced rabbit reticulocytes was studied with the non-metabolized folate-analog, methotrexate. The time-course of methotrexate uptake into a mixed population of reticulocytes and mature erythrocytes is a two-component process consisting of a small, but rapid, initial uptake phase followed by a much slower uptake component which remains essentially constant over the period of observation. The velocity of the latter uptake component is directly proportional to the per cent reticulocytes and appears to represent a unidirectional influx of methotrexate into these cells. Uptake of methotrexate into reticulocytes was found to have the following characteristics: (a) temperature sensitivity, Q₁₀ of 4; (b) uptake velocity as a function of the extracellular methotrexate concentration approximated Michaelis-Menten kinetics with a maximum transport velocity of 48 pmoles/min per g dry wt; the extracellular methotrexate level at which the uptake velocity was one-half maximum was 1.4 μM; (c) 5-formyltetrahydrofolate markedly inhibited methotrexate uptake but pteroylglutamic acid inhibition was weak; (d) uptake was stimulated in cells preincubated with 5-formyltetrahydrofolate, indicative of hetero-exchange diffusion; (e) uptake was independent of extracellular sodium but was inhibited by anions including nitrate, phosphate, and glucose-6-phosphate; (f) uptake was enhanced by azide plus iodoacetate.These data indicate that folate transport in rabbit reticulocytes is mediated by a carrier mechanism which disappears with reticulocyte maturation. The mechanism of folate transport in rabbit reticulocytes is qualitatively similar to tumor cells previously studied; both appear to have an energy-dependent mechanism limiting folate uptake, and influx in both is inhibited by structurally unrelated inorganic and organic anions. These studies suggest that circulating pteroylglutamic acid is of little importance in meeting the folate requirements of folate-dependent tissues and raise the possibility that clinical conditions associated with alterations in the anionic composition of the blood may be accompanied by impaired utilization of the folates.