A high-performance liquid chromatography assay was developed for the purpose of examining xanthine, hypoxanthine, uridine, thymidine, allopurinol, and oxypurinol in serum of patients with advanced carcinoma treated with methorexate and 5-fluorouracil. After the addition of an internal standard, serum samples were extracted of purines and pyrimidines with trichloroacetic acid and neutralized withtert-amine. Aliquots of the extracted serum were injected onto a C18 reverse-phase column and purines and pyrimidines were eluted with a gradient of MeOH/H2O and KH2PO4solutions. Absorbance was detected with a variable-wavelength UV spectrophotometer at 254 nm and 280 nm. This assay can be readily applied to quantitate baseline and treatment-induced variations in serum purine and pyrimidine levels which may correlate with clinical response and/or toxicity in patients with cancer.