F2-MTX(r)A is an L1210 leukemia cell line with a functional defect in the reduced folate carrier and high level expression of folate receptor β. The pH-dependence of methotrexate (MTX) influx by folate receptor β in F2-MTX(r)A cells was characterized and compared with that of the reduced folate carrier in parental L1210 cells. MTX influx by folate receptor β had a pH optimum of 6.5, whereas influx mediated by the reduced folate carrier showed a pH optimum of 7.5. Increased folate receptor β-mediated MTX influx at pH 6.5 relative to pH 7.5 was accompanied by a 5-fold increase in binding affinity of the receptor for MTX without a change in the number of binding sites. At pH 6.2, approximately 24% of MTX influx in F2-MTX(r)A cells proceeded by another mechanism. This transport route became active at pH <7.5, operated optimally at pH 6.0 to 6.5, and, unlike folate receptor β-mediated MTX influx, was insensitive to the presence of low levels of folic acid (100 nM). MTX influx by the low pH system showed saturability, with a K(t) of 5.3 μM and a V(max) of 1.53 nmol/g dry wt/min, was energy dependent, was inhibited by sulfobromophthalein with a K(i) of 148 μM, and had similar relative affinities for folic acid, leucovorin, and 5-methyltetrahydrofolate. Influx of 5-methyltetrahydrofolate was also mediated by this route. The data provide further confirmatory evidence for an MTX influx route in F2-MTX(r)A cells, optimal at low pH and distinct from the reduced folate carrier or the folate receptor.