Various independent kinetic criteria for indicating multiplicity of carrier-mediated entry of folate compounds into L1210 cell plasma membrane vesicles are studied. We find a marked inconsistency between values for influx Km and Ki in reciprocal experiments measuring competition between various folate compounds as well as inconsistent effects of transloading shown for 5-formyltetrahydrofolate influx, but not folic acid influx. These results argue strongly against a one-carrier model for transport of folate compounds. The most straightforward interpretation of our data is that two distinct transport systems mediate entry of folate compounds in L1210 plasma membrane vesicles. If a two-carrier model is correct, then our data indicate that one of the carriers has low capacity and high affinity for folate coenzymes and methotrexate. This system is apparently negligible as a transport route for folic acid. Transtimulation of initial influx by substrates of the low capacity system is obtained following transloading with coenzymes but not by transloading with folic acid. Our data indicate that the second folate transport system postulated by the two-carrier model has a low affinity for all the folate compounds studied. Nevertheless, the putative second system is significant, especially for folic acid transport, because it has a much higher capacity than the first transport system. In contrast to the first system, transloading with any of the folate compounds studied had no effect on initial influx mediated by the second folate transport system. The two systems are also differentially inhibited by pCMBS, DIDS and SITS and the influx Vmax for the high-affinity/low-capacity system was altered in a vesicle preparation derived from a methotrexate resistant L1210 cell line.