A new fluorescent methotrexate analogue (PT430) was synthesized as a reported ligand for dihydrofolate reductase. The analogue was prepared by attachment of lysine in place of the glutamate side chain of methotrexate and conjugation to fluorescein isothiocyanate via the epsilon-amino group of lysine. Spectrophotometric enzyme inhibition assays showed PT430 to be about one-tenth as potent as methotrexate against either Lactobacillus casei or L1210 mouse leukemia enzyme; competitive radioligand binding assays using tritiated methotrexate gave similar results. In assays of L1210 cell proliferation in culture, on the other hand, PT430 was 100-fold less toxic than methotrexate. In dilute solution, the fluorescence intensity of PT430 was 5-fold lower than that of equimolar fluorescein and diminished with decreasing pH. On complexation with dihydrofolate reductase, however, fluorescence intensity was enhanced 3- to 5-fold depending on the pH. Measurement of fluorescence increase with added ligand provided data for the determination of the stoichiometric ratio, dissociation constant, and extent of fluorescence enhancement. Specificity of PT430 for methotrexate binding sites was indicated by the observation of decreased fluorescence uptake in PT430-treated L1210 cells in the presence of methotrexate. Fluorescence uptake occurred faster, and to a greater extent, in methotrexate-resistant dihydrofolate reductase overproducing L1210/R6 cells than in the methotrexate-sensitive parent line. Therefore, PT430 may be used as a flow cytometry probe to detect methotrexate resistance based on dihydrofolate reductase overproduction.