The uptake of 2',3'-dideoxyadenosine was examined in a human immunodeficiency virus (HIV) infected and uninfected T cell line (H9 cells), a B cell line (Namalwa), and in normal peripheral blood mononuclear cells. After a 10-minute incubation at ambient temperature, the intracellular 2',3'-dideoxyadenosine-derived radioactivity was 8- to 16-fold higher than the extracellular radioactivity. In metabolically inactive cells (0 degrees C), the intracellular and extracellular 2',3'-dideoxyadenosine-derived radioactivities were nearly equal. In infected and noninfected H9 cells, a large excess of p-nitrobenzylmercaptopurine riboside or pyrimidine nucleosides weakly inhibited the uptake of 2',3'-dideoxyadenosine (7-30%), whereas deoxycoformycin was a stronger inhibitor (50-80%). Purine nucleosides minimally enhanced the uptake (10-20%). The cellular uptake was not associated with the accumulation of dideoxyadenosine triphosphate. In normal peripheral blood mononuclear cells, the uptake of 2',3'-dideoxyadenosine was inhibited by all agents except 2'-deoxyadenosine (15% enhancement). In contrast to H9 cells, the formation and accumulation of dideoxyadenosine triphosphate paralleled the uptake of dideoxyadenosine. The results of these studies suggest that the major route of transport of 2',3'-dideoxyadenosine into cells is by simple diffusion and that different metabolic patterns exist among cell lines and normal peripheral blood mononuclear cells. An understanding of these cellular differences could aid in the development of therapeutic strategies directed against HIV.