Intravenous methotrexate (MTX) therapy is widely used for treatment of various neoplastic diseases in children. The optimization of the MTX dose and/or the subsequent leucovorin rescue is based on pharmacokinetic data calculated from plasma concentrations collected after cessation of the MTX administration. The influence of the MTX assay method on the subsequent pharmacokinetic evaluation was studied in 13 children with acute lymphoblastic leukemia. Plasma samples were collected after administration of MTX (5-8 g/m2) as 24 h infusions. All samples were analyzed by five different analytical procedures, viz. liquid chromatography (LC), enzyme inhibition assay (EIA), two fluorescence polarization immunoassays (FPIA1 and FPIA2) and enzyme multiplied immunoassay (EMIT). Using measurements from the four non-chromatographic procedures, only about 50% of determined pharmacokinetic parameters (area under the plasma concentration time curve, calculated by the trapezoidal rule and from pharmacokinetic modelling, and the terminal half life time) were within the range 75-125% of the values obtained from LC data. We conclude that the clinical outcome of MTX therapy using estimated MTX pharmacokinetics as guidelines for proper dosing of MTX and/or leucovorin rescue might be affected by the lack of accuracy of non-chromatographic procedures for MTX analysis. There is still a need for improving the accuracy of the procedures aimed at therapeutic drug monitoring of MTX.