10-Ethyl-10-deaza-aminopterin (10-EdAM) is a novel folic acid antimetabolite currently being tested in phase II clinical trials. We have developed an isocratic high-performance liquid chromatographic method for the quantification of 10-EdAM and metabolites in plasma. Solid-phase extraction was used for sample clean-up. Adequate accuracy was obtained without the use of an internal standard. Fluorometric detection with excitation at 243 nm and emission at 488 nm was used for accurate quantification of samples containing small amounts of drug or metabolites (2.0-4.0 nM, depending on the compound). Ultraviolet detection at 350 nm was only applicable for the analysis of plasma concentrations of 10-EdAM exceeding 50 nM. The usefulness of the assay was demonstrated by the results obtained in a pharmacokinetic study. The assay could separate the parent compound from seven identified and two unknown products.