1954U89, 1,3-diamino -7 - (1-ethylpropyl) -8- methyl-7H-pyrrolo(3,2-f)-quinazoline, is a potent, lipid-soluble inhibitor of dihydrofolate reductase that is under preclinical evaluation as an anticancer agent. A rapid and selective high performance liquid chromatographic assay with fluorescence detection was developed for the quantitation of 1954U89 in rat and dog plasma. The compound was removed from plasma by solid phase extraction, and the extracts were chromatographed on a Hypersil C1column (4.6 mm × 15 cm) under isocratic conditions. The HPLC mobile phase consisted of methanol and 0.02 M ammonium acetate buffer (pH = 4.5) delivered in a ratio of 70:30 and at a flow rate of 1.0 mL/min. The compound was quantitated by fluorescence detection with excitation and emission wavelengths set at 335 and 460 nm, respectively. The quantitation range of the assay was 0.01 to 2.0 μg/mL. The intra- and interassay precision of the method were approximately 4 and 6%, respectively, in rats, and approximately 7 and 4%, respectively, in dogs. The accuracy (% bias) ranged from−10to+2% across the concentration range in both species. This assay has been used to support nonclinical pharmacokinetic and bioavailability studies of 1954U89 in rats and dogs.