The antifolate compounds IOdeazaaminopterin (lOdAM) and IO-ethyl-IOdeazaaminopterin ( IO-EdAM) are therapeutically superior to methotrexate in transplanted murine tumor systems and in human tumor xenografts growing in immunodeficient "nude" mice. The increased therapeutic index of these analogs correlates with their selective uptake, retention, and polyglutamation within neoplastic cells. We have developed a Iluorescence high-performance liquid chromatographic assay applicable to IO-dAM, IO-EdAM, their polyglutamate anabolites, and their 7-hydroxy (7-OH) and deglutamate catabolites. The assay is based upon the high native fluorescence of pteridine-containing compounds which contain carbon in the 10 position. The assay employs a reverse-phase C-l 8 column and an ascending acetonitrile gradient in 50 mM phosphate, pH 7.0. The compounds are extracted from plasma and urine with 95 * 7% and 98 + 2% recoveries. respectively, using C-18 SepPaks. The linear range of the assay is, for 1 O&AM, 2-100 nM, and for I 0-EdAM, 1 -100 nM. Polyglutamated metabolites of [3H] IO-EdAM isolated from Ll2 10 cells have been separated by HPLC with identification of five derivatives (Glu l-5) confirmed by enzymatic peak shift using serum conjugase and by quantitative correlation of fluorescence intensity, radioactivity, and titration inhibition of dihydrofolate reductase. The assay has been used successfully in pharmacokinetic analyses of plasma and urine samples from patients receiving IO-dAM and lo-EdAM. In patients who had received IO-EdAM, 7-OH-IO-EdAM, and the deglutamate catabolite were also detected. This HPLC fluorescence assay is superior to the dihydrofolate reductase inhibition and binding assays with regard to specificity and precision; moreover, it can provide a means for simultaneous assay of the physiologically important anabolites and catabolites of these new antifolates. Q 1985 academic press, IX.