Prior studies from this laboratory documented the prevalence of methotrexate (MTX) transport activity with a low pH optimum in human solid tumor cell lines. In HeLa cells, this low pH activity has high affinity for pemetrexed [PMX (Alimta)] and is reduced folate carrier (RFC)-independent because it is not diminished in a RFC-null subline (R5). R5 cells also have residual transport activity, with high specificity for PMX, at neutral pH. In the current study, a R5 subline, R1, was selected under MTX selective pressure at a modest reduction in pH. There was markedly decreased MTX and PMX transport at both pH 5.5 and pH 7.4. When MTX was removed, there was a slow return of transport activity, and when MTX was added back, there was loss of transport at both pH values within 8 weeks. In R1 cells, there was a marked decrease in accumulation of PMX, MTX, and folic acid along with a decrease in growth inhibition by these and other antifolates that require a facilitative process to gain entry into cells. These data demonstrate that (i) RFC-independent transport in HeLa cells at low and neutral pH contributes to antifolate activity (in particular, to PMX activity) and can be diminished by antifolate selective pressure and (ii) the loss of these activities results in marked resistance to PMX, an agent for which there is little or no loss of activity when transport mediated by RFC is abolished. These observations suggest that transport activity in RFC-null HeLa R5 cells at neutral and low pH may reflect the same carrier-mediated process.