The Chinese hamster lung cell line DC-3F8/A55 has a 4,500-fold increase in resistance to methotrexate over the parental cell line DC-3F. Although DC- 3F8/A55 cells have a 4.5-fold increase in a mutant form of dihydrofolate reductase, this does not fully account for the high level of resistance to methotrexate. The purpose of this study is to determine the molecular basis for the inability of DC-3F8/A55 cells to accumulate methotrexate, and to identify the mechanism of transport of folate compounds in DC-3F8/A55 cells. This work has revealed that DC-3F8/A55 cells harbor a debilitating mutation to the reduced folate carrier gene, resulting in the loss of reduced folate carrier function. A nonsense mutation changes an arginine at amino acid 88 to a STOP codon, resulting in a non-functional protein. The parental cell line DC-3F is heterozygous at this locus, possessing one mutant and one wild-type allele of the RFC gene, thus retaining reduced folate carrier activity. These facts are supported by the kinetics of folate transport in both of these cell lines. The parental cell line DC-3F has a Kt for folinic acid of 10.69 +/- 0.67 muM and for methotrexate of 8.88 +/- 0.82 muM, values characteristic of a cell expressing a reduced folate carrier. DC-3F8/A55 cells were found to have a Kd for MTX of 3.16 +/- 1.03 nM, for folinic acid of 7.75 +/- 2.16 nM, and for folic acid of 1.42 +/- 0.54 nM. The high affinity of DC-3F8/A55 cells for folic acid, with a Kd for folic acid in the nM range, suggests that these cells are expressing a folate receptor. Northern blot analysis revealed a 1.6 kb transcript with low homology to FR-alpha and FR-gamma in DC-3F8/A55 cells. Overall, these studies suggest that the methotrexate transport-defective cell line DC-3F8/A55 expresses a previously unidentified folate receptor which may be a new member of the folate receptor family.