The antifolate methotrexate (MTX) is widely used in cancer chemotherapy. In this study, we show that MTX (MTX-Glu(1)) and MTX-polyglutamates (MTX-Glu(2-5)) strongly inhibited the growth of the leukemic cell line MOLT-4. This effect, however, was mitigated by ascorbic acid. We investigated whether ascorbic acid is able to reduce dihydrofolic acid (DHF) to tetrahydrofolic acid (THF) directly or by circumventing the MTX inhibition of dihydrofolate reductase (DHFR). The inhibition of this NADPH-dependent reduction of DHF by MTX-Glu(n) in the absence or presence of ascorbate, was determined by analytical isotachophoresis. Using 0.01 M HCl/histidine, pH 6.0, as a leading electrolyte (L) and 0.005 M 2-(N-morpholino)ethanesulfonic acid (MES)/histidine, pH 6.0, as a terminating electrolyte (T), MTX-Glu(n) derivatives including MTX-Glu(1) could be easily separated, whereas the quantitative estimation of THF was not possible. A quantitative characterization of the DHFR reaction by measuring NADPH, NADP(+) and ascorbate was achieved with another system (L: 0.01 M HCl/beta-alanine, pH 3.73; T: 0.01 M caproic acid, pH 3.27). Nanomolar concentrations of MTX-Glu(1-5) inhibited consumption of NADPH and production of NADP(+). Ascorbic acid was not able to reduce DHF, neither directly nor after inhibition of DHFR by MTX. However, ascorbic acid seemed to diminish the oxidation of THF and this may account for its capacity to reduce the inhibitory effect of MTX on MOLT-4 cells.