A phenylalanine substitution for serine in the reduced folate carrier at residue 309 (RFC1-S309F) was identified in a methotrexate (MTX)-resistant cell line selected with 5-formyltetrahydrofolate (5-CHO-THF) as the sole folate source. The transport characteristics of the mutated carrier were studied by transfection into the MTXrA line, which lacks endogenous RFC1 function. The level of expression of carrier in the cell lines studied was determined by specific surface binding of 5-methyltetrahydrofolate (5-CH3-THF). Influx of 5-CH3-THF and 5-CHO-THF mediated by RFC1-S309F was 20- and 7-fold greater than that of MTX, respectively. Consistent with the influx difference between 5-CHO-THF and MTX, the growth requirement (EC50) for 5-CHO-THF in MTXrA-S309F cells was decreased by a factor of 9, while the MTX IC50 was reduced by a factor of only approximately 2 as compared with the recipient MTXrA cells. The decrease in 5-CH3-THF influx mediated by the mutated carrier was attributed to a decrease in the mobility of the 5-CH3-THF-carrier complex, since the influx Kt was essentially unchanged. However, the reduction in 5-CHO-THF and MTX influx was attributed to decreases in both carrier affinity and Vmax, although the decline in the MTX influx Vmax appeared to be much greater than for 5-CHO-THF. The inhibitory effect of chloride on 5-CHO-THF influx observed for L1210 cells was eliminated in the MTXrA-S309F line. This study represents another example of a single mutation in RFC1 that markedly impairs MTX influx but partially preserves transport of reduced folates when cells are selected with 5-CHO-THF as the available folate substrate. The data indicate that residues in the predicted eighth transmembrane domain of RFC1 can play an important role in the selectivity of folate binding and the mobility of the carrier-substrate complex.