The disposition and metabolic fate of 3'-deoxy-2',3'-didehydrothymidine (D4T) were evaluated both in isolated hepatocytes and in nonhuman primates. Rapid formation of thymine and beta-aminoisobutyric acid (BAIBA) occurred following incubation of hepatocytes with 10 microM [5(-3)H]D4T. Substantial levels of tritiated water were also detected. Exposure of cells to D4T in the presence of either 1 mM thymine or 10 microM benzyloxybenzyluracil, an inhibitor of dihydropyrimidine dehydrogenase, decreased intracellular BAIBA levels by approximately 89 and 63%, respectively. Concurrently, [3H]thymine levels increased two- to fivefold. These results are consistent with D4T being cleaved to thymine, which is then degraded to BAIBA. A similar metabolic disposition was observed in monkeys following administration of 25 mg of [5(-3)H]D4T per kg of body weight. BAIBA, thymine, and tritiated water were identified in plasma and urine. Approximately 50% of the administered dose was recovered in urine within 24 h, with the majority of the radioactivity representing unchanged drug. After administration intravenously or orally of 25 mg of [4(-14)C]D4T per kg of body weight to monkeys, a novel metabolite, designated X, in addition to unchanged D4T, thymine, and BAIBA, was also detected. The sum of the three metabolites and unchanged drug accounted for virtually all of the radioactivity in plasma and urine. Thymine and X exhibited kinetic profiles similar to that of D4T, with plasma elimination half-life of 2 to 3 h, whereas BAIBA levels remained constant for extended periods and declined slowly; this metabolite could be detected 24 h after intravenous drug administration. Mean oral bioavailability of D4T was high at approximately 70%. As observed in the [5(-3)H]D4T study performed in monkeys, approximately half of the administered [4(-14)C]D4T was recovered unchanged. The remainder was not recovered in urine or feces collected up to 30 days after drug administration. These data suggest that D4T metabolites are further metabolized by salvage pathways and/or converted to biological macromolecules.