Radiolabeled caffeine was administered orally at 5 mg/kg to adult, male volunteers. Blood, saliva, expired CO2, urine, and feces were collected and analyzed for total radiolabeled equivalents, caffeine, and its metabolites. High-performance liquid chromatography (HPLC) was the principal technique used to separate caffeine and the various metabolites with quantitation by liquid-scintillation counting. The half-life of caffeine in both serum and saliva was approximately 3 hr, with the concentration of caffeine in the saliva samples ranging from 65 to 85% of that found in the serum samples. The major metabolites found in serum and saliva were the dimethylxanthines. In the course of separating the urinary metabolites, our HPLC system partially resolved two unidentified polar metabolites arising from radiolabeled caffeine. The major component corresponded to 5-acetylamino-6-amino-3-methyluracil and in our subjects ranged from 7 to 35% of the administered dose. The other principal urinary metabolites were 1-methylxanthine at approximately 18% of the administered dose and 1-methyluric acid at 15%. The fecal samples contained approximately 5% of the dose, mainly as uric acid compounds which retained the 1-methyl group. In this study we accounted for approximately 90% of the administered radiolabeled dose and identified greater than 95% of the urinary radioactivity as specific metabolites.