A rapid and sensitive liquid chromatographic assay for 2',3'-didehydro-3'-deoxythymidine (d4T) in plasma and urine is described. This assay uses thymidine oxetane (TO), a synthetic precursor of d4T, as internal standard. Sample preparation involves a simple extraction of plasma or urine with 5% isopropyl alcohol in methylene chloride. The method is specific and sensitive, allowing a linear response over a 2000-fold range of concentrations in human plasma (5 ng/ml to 10 micrograms/ml) and urine (50 ng/ml to 100 micrograms/ml). This assay, developed for human plasma and urine, is also applicable to rabbit samples with minor modification. Intravenous bolus doses of 10 mg/kg d4T to rabbits showed that the plasma concentration-time profile followed a biexponential decay. Estimates of the distribution and elimination half-lives were 6.7 +/- 0.9 and 51 +/- 6 min, respectively. The total-body and renal clearances were 23.4 +/- 3.6 and 8.82 +/- 3.9 ml/min.kg, respectively. That the renal clearance exceeds the glomerular filtration rate in the rabbit suggests that d4T is actively secreted in the renal tubule. The fraction excreted unchanged in the urine was 36 +/- 8%. Similar results were obtained in the same rabbits at steady state during constant-rate intravenous infusion. Noncompartmental analysis estimates of the MRT and Vdss were 46 +/- 5 min and 1.08 +/- 0.13 L/kg, respectively.