[1-[2',5'-Bis-O-(tert-butyldimethylsilyl)-beta-D-ribofuranosyl]-3-N- methyl-thymine]-3'-spiro-5''-(4''-amino-1'',2''-oxathiole-2'',2''- dioxide) (TSAO-m3T) is a potent, selective and specific inhibitor of human immunodeficiency virus type 1 replication in vitro. Uptake of TSAO-m3T by human CEM cells is drug concentration-dependent and increased proportionally with increasing initial extracellular TSAO-m3T concentrations up to 20 micrograms/mL. Within 6 hr of incubation, the cells were almost completely saturated with the test compound; further incubation up to 72 hr did not markedly increase the intracellular concentration of the compound. No intracellular metabolic conversion of TSAO-m3T was observed in CEM, MT-4 or MOLT-4 cells. Upon intravenous bolus administration of TSAO-m3T to mice at 0.75 mg/kg, TSAO-m3T was rapidly cleared from the plasma in a mono-exponential manner (half-life: 22 min; distribution volume: 9.5 L/kg; total body clearance: 17.8 L/hr/kg). TSAO-m3T mainly accumulated in the lungs, followed by the heart, kidney and liver. Significant amounts of different metabolites of TSAO-m3T were detected in most tissues, the liver, kidney and spleen being the organs that showed the most extensive metabolism. The principal metabolites identified were TSAO-m3T derivatives in which the t-butyldimethylsilyl moiety at C-2' and/or C-5' had been split off. The free base N3-methylthymine was not detected.