Several mechanisms have been described to explain the resistance of cells to methotrexate (MTX); however, the basis for the heterogeneity of mechanisms has been obscure. It was hypothesized that the type of MTX resistance in a single species can be influenced by the form of extracellular folate supplied during the development of resistance. Two strains of MTX-resistant Enterococcus faecium were developed by transferring the bacteria to media containing increasing concentrations of MTX in the presence of constant concentrations of either 5-formyl-5,6,7,8-tetrahydropteroylglutamic acid (5-HCO-H4PteGlu) or pteroylglutamic acid (PteGlu). These resistant strains were designated E. faecium/MTX/5-HCO-H4PteGlu and E. faecium/MTX/PteGlu, respectively. The mechanisms of MTX resistance included: (1) increased folic acid reductase (FAR) activity in both resistant strains but increased dihydrofolate reductase (DHFR) activity only in E. faecium/MTX/PteGlu; (2) decreased synthesis and intracellular retention of MTX containing two glutamyl residues; (3) decreased uptake of MTX accompanied by decreased uptake of folates; and (4) reduction of folate-binding capacity. Among these, the form of folate present in the media during the development of resistance affected DHFR and FAR activities and the transport of folates. These findings, together with data from other laboratories, suggest that it may be important to use a reduced form of folate, a more physiological form than oxidized PteGlu, in the media during the development of resistance for the study of the mechanisms of MTX resistance in cultured cells.