The reduced folate carrier (RFC1) is a major transporter for both natural reduced folates and antifolate chemotherapeutics. Using polyclonal antibodies targeted to epitopes at the loop between the sixth and seventh predicted transmembrane domains or the distal C-terminus, we were able to demonstrate by Western blot analysis that the molecular size of RFC1 expressed in murine leukemia L1210 cells is 58 kDa as predicted by the open reading frame of its cDNA. 46- and 38-kDa proteins detected only in plasma membrane preparations were proteolytic degradation products that appeared during membrane preparation or treatment with the conventional SDS-PAGE loading buffer. These data resolve discrepancies reported previously for the molecular size of RFC1.