The intracellular metabolism of the -L- enantiomer of 2',3'-dideoxyadenosine (-L-ddA) was investigated in HepG2 cells, human peripheral blood mononuclear cells (PBMC), and primary cultured human hepatocytes in an effort to understand the metabolic basis of its limited activity on the replication of human immunodeficiency virus and hepatitis B virus. Incubation of cells with 10 µM [2',3',8-³H]--L-ddA resulted in an increased intracellular concentration of -L-ddA with time, demonstrating that these cells were able to transport -L-ddA. However, it did not result in the phosphorylation of -L-ddA to its pharmacologically active 5'-triphosphate (-L-ddATP). Five other intracellular metabolites were detected and identified as -L-2',3'-dideoxyribonolactone, hypoxanthine, inosine, ADP, and ATP, with the last being the predominant metabolite, reaching levels as high as 5.14 ± 0.95, 8.15 ± 2.64, and 15.60 ± 1.74 pmol/10⁶ cells at 8, 4, and 2 h in HepG2 cells, PBMC, and hepatocytes, respectively. In addition, a -glucuronic derivative of -L-ddA was detected in cultured hepatocytes, accounting for 12.5% of the total metabolite pool. Coincubation of hepatocytes in primary culture with -L-ddA in the presence of increasing concentrations of 5'-methylthioadenosine resulted in decreased phosphorolysis of -L-ddA and formation of associated metabolites. These results indicate that the limited antiviral activity of -L-ddA is the result of its inadequate phosphorylation to the nucleotide level due to phosphorolysis and catabolism of -L-ddA by methylthioadenosine phosphorylase (EC 2.4.2.28).