The properties of the folate transport system in H35 hepatoma cells have been studied by measuring the transport of (+)-5-methyltetrahydrofolate. Using initial rates of uptake, it has been demonstrated that the uptake is saturable, carrier mediated, and shared by methotrexate. The accumulation of (+)-5-methyltetrahydrofolate is concentrative, demonstrating the presence of an active transport process. A previous study suggested that methotrexate-resistant sublines (H35R) acquired methotrexate insensitivity because of an impaired capacity for transport. This postulate was substantiated in the present investigation by several observations. The initial uptake and steady-state level of (+)-5-methyltetrahydrofolate were markedly reduced in the resistant sublines as was the case with methotrexate. Triazinate (2-(chloro-4-[4,6-diamino-2,2-dimethyl-S-triazine-1(2H)-ylphenoxyl])-N,N-dimethyl-m-toluamide . ethanesulfonic acid) an inhibitor of dihydrofolate reductase which enters the cells by a pathway independent of the folate coenzyme, was equally toxic to H35 cells and to an H35 subline resistant to 0.3 microM methotrexate. Resistant sublines that are insensitive to methotrexate up to 1 microM display a transport defect but have normal levels of dihydrofolate reductase. Sublines resistant to higher levels of methotrexate showed not only defective transport but also commensurate increases in dihydrofolate reductase. Attempts to demonstrate carried-dependent transport of (+)-5-methyltetrahydrofolate or methotrexate in resistant sublines were negative, suggesting the lack of a functional carrier. These properties were readily demonstrated in H35 cells and included temperature dependence, competition for uptake with analogs, and transstimulation.