The antiviral activity, uptake, and metabolism of 2',3'-dideoxyguanosine was investigated in human immunodeficiency virus- (HIV) infected and noninfected human cells. 2',3'-Dideoxyguanosine had anti-HIV activity (effective dose 50%: 0.1-1.0 microM) in H-9 and MT-2 cells. The addition of excess (greater than or equal to 30 microM) guanosine, deoxyguanosine, or 8-aminoguanosine had no effect on the anti-HIV activity of 2',3'-dideoxyguanosine. In [8-3H]2',3'-dideoxyguanosine-exposed cells, the intracellular radioactivity was twofold higher than the extracellular. When guanosine, deoxyguanosine, or 8-aminoguanosine was preincubated or added simultaneously to 2',3'-dideoxyguanosine, uptake of 2',3'-dideoxyguanosine was reduced by 28 to 34%, whereas addition of p-nitrobenzylmercaptopurine riboside (20 microM) had no effect. In metabolism studies using H-9 cells, dideoxyguanosine triphosphate could not be detected despite a 24-h incubation of 2',3'-dideoxyguanosine at effective anti-HIV concentrations. The addition of excess (greater than or equal to 30 microM) guanosine, deoxyguanosine, and 8-aminoguanosine, while inhibiting the catabolism of 2',3'-dideoxyguanosine, did not enhance the anabolic conversion of 2',3'-dideoxyguanosine to dideoxyguanosine triphosphate. Our failure to detect the formation of dideoxyguanosine triphosphate and the lack of reversal of antiviral effects by natural purine nucleosides raises questions on the role of this metabolite in the anti-HIV activity of 2',3'-dideoxyguanosine.