The folate antagonist methotrexate is a cytotoxic drug used in the treatment of several cancer types. The entry of methotrexate into the cell is mediated by two main transport systems: the reduced folate carrier and membrane-associated folate receptors. These transporters differ considerably in their mechanism of (anti)folate uptake, substrate specificity, and tissue specificity. Although the mechanism of action of the reduced folate carrier is fairly well-established, that of the folate receptor has remained unknown. The development of specific folate receptor-targeted antifolates would be accelerated if additional mechanistic data were to become available. In this work, we used two fluorescently labeled conjugates of methotrexate, differently linked at the terminal groups, to clarify the uptake mechanism by folate receptor-α. The results demonstrate the importance of methotrexate amino groups in the interaction with folate receptor-α.