A broad spectrum of structurally diverse anions reversibly inhibits the influx of methotrexate in L1210 cells. Several of the more effective anions and their respective inhibition constants (Ki values) were: 5-methyltetrahydrofolate (0.3 microM), bromosulfophthalein (2 microM), thiamine pyrophosphate (3 microM), 8-anilino-1-naphthalene sulfonate (7 microM), phthalate (20 microM), and AMP (50 microM). Moderate inhibition was observed with Pi (Ki = 400 microM) and other divalent inorganic anions, while small monovalent anions such as Cl- (Ki = 30 mM) were the least effective. When these same anions were tested for an effect on methotrexate efflux, stimulation was observed with some anions, while others had no effect. Enhancement was produced by folate compounds and p-amino-benzoylglutamate, small monovalent (e.g., Cl-, acetate, and lactate) and divalent (e.g., phosphate and succinate) anions, a few nucleotides (e.g., AMP), and thiamine pyrophosphate, while little or no effect was associated with trivalent anions (e.g., citrate), most nucleotides, and large organic anions (e.g., bromosulfophthalein, NAD, and NADP). Anions with the ability to promote methotrexate efflux in control cells lost this capacity upon exposure of the cells to an irreversible inhibitor of methotrexate influx. These results support the hypothesis that methotrexate transport proceeds via an anion-exchange mechanism and moreover provide evidence that anion substrates for this system can be identified by their ability to promote methotrexate efflux. Anions which appear most likely to participate in this exchange cycle in vivo are Pi and AMP.