A human reduced folate carrier (hRFC) cDNA was transfected into transport-deficient K562 cells to circumvent complications that may result from carrier expression in a heterologous mammalian species. Relative to wild-type cells, hRFC transcript levels were increased 11- and 19-fold, respectively, in the K43-6 and K43-1 transfectants. Although photoaffinity labeling of hRFC protein revealed similar increases of 15- and 19-fold, respectively, only a 2-fold enhancement in methotrexate (Mtx) transport was observed. This suggests that only a small portion of the cDNA-encoded hRFC protein is actively engaged in membrane transport. Kinetic analysis of [3H]Mtx transport indicated that K43-6 cells exhibited a similar affinity (Kt) but an increased Vmax (1.7-fold) when compared with K562 cells. The restored transport was similar to that of wild-type cells in its capacity to be trans-stimulated by intracellular folates and in its sensitivity to competitive transport inhibitors (1843U89, bromosulfophthalein, folic acid, leucovorin, and ZD1694) and to irreversible inhibition by N-hydroxysuccinimide-methotrexate. Further, deglycosylated photoaffinity-labeled hRFC protein in both K562 and K43-6 cells migrated at approximately 65-70 kDa on SDS-gels, consistent with the molecular mass from the predicted amino acid sequence. These data further establish that the expression of hRFC, alone, is sufficient to confer transport properties typical of the "classical" hRFC. However, the discrepancy between the stoichiometry of carrier expression and transport activity implies that membrane translocation of bound substrate may be regulated by additional undefined mechanisms.