An automated high-performance liquid chromatographic system is described for separation and quantitation of the antineoplastic drug methotrexate and metabolites, and the antibiotic sulfamethoxazole in body fluids. The 40-min analysis utilizes a reversed-phase C18 column and gradient elution with detection by absorbance of ultraviolet light at 308 nm. The minimum detectable quantities with this assay are: methotrexate 4.4 ng (9.8 X 10(-12) mole); 4-deoxy-4-amino-N10-methylpteroic acid 11.9 ng (3.7 X 10(-11) mole); 7-hydroxymethotrexate 30 ng (6.5 X 10(-11) mole); sulfamethoxazole 125 ng (4.9 X 10(-10) mole). This analytical method should prove useful for therapeutic monitoring and pharmacokinetic studies of these compounds.