Sensitive high-performance liquid chromatographic assays have been developed for the quantification of stavudine (2',3'-didehydro-3'-deoxythymidine, d4T) in human plasma and urine. The methods are linear over the concentration ranges 0.025-25 and 2-150 microg/ml in plasma and urine, respectively. An aliquot of 200 microl of plasma was extracted with solid-phase extraction using Oasis cartridges, while urine samples were simply diluted 1/100 with HPLC water. The analytical column, mobile phase, instrumentation and chromatographic conditions are the same for both methods. The methods have been validated separately, and stability tests under various conditions have been performed. The detection limit is 12 ng/ml in plasma for a sample size of 200 microl. The bioanalytical assay has been used in a pharmacokinetic study of pregnant women and their newborns.