For the determination of tetrahydrofolate (H_4folate) and 5-methyltetrahydrofolate (5-CH_3-H_4folate), a high-performance liquid chromatography (HPLC) system composed of a single reversed-phase octadecylsilane column and fluorescence detector by an isocratic solvent was proposed. This HPLC system perfectly analyzed H_4folate and 5-CH_3-H_4folate in biological samples. We applied this HPLC system into assays of methylenetetrahydrofolate reductase and methionine synthase activities by monitoring their products of 5-CH_3-H_4folate and H_4folate, to give a new method for determining the enzyme activity without use of radioactive compounds, and the sensitivity of the method is sufficient for measurement of these enzyme activities in crude extracts. Using sodium ascorbate and 2-mercaptoethanol or dithiothreitol,folate derivatives and enzymes were stable in the assay process. No reagents in the assay mixture were found to disturb the separation and detection of both 5-CH_3-H_4folate and H_4folate in our assay system. In addition, our analytical procedure is very simple and economical.