Four routine assays commonly used for monitoring plasma methotrexate (MTX) during high-dose therapy were validated by HPLC as the comparison method. MTX and its main metabolite, 7-hydroxymethotrexate (7-OHMTX), were analyzed by HPLC with postcolumn derivatization and fluorometric detection. About 200 clinical plasma samples from 13 children with acute lymphoblastic leukemia who received 5-8 g/m2 MTX as 24-h infusions were analyzed. The fraction of measured concentrations of MTX that were within 75-125% of the values obtained by HPLC were 64.5% for enzyme inhibition assay, 56.4% for fluorescence polarization immunoassay with polyclonal antibodies (FPIA1; Abbott), 58.9% for FPIA2 (with monoclonal antibodies; Abbott), and 46.4% for enzyme-multiplied immunoassay (Emit; Syva). All nonchromatographic procedures were subject to interferences from MTX plasma metabolites or endogenous substances. The interference from 7-OHMTX was, however, somewhat less pronounced for FPIA2 (monoclonal) than for FPIA1 (polyclonal).