4'-Azidothymidine (ADRT) is a novel nucleoside analogue that exhibits potent inhibitory activity against the replication of human immunodeficiency virus (HIV) in lymphocytes. The mechanisms by which ADRT inhibits HIV reverse transcriptase (HIV-RT) as ADRT 5'-triphosphate (ADRT-TP), the active intracellular metabolite of ADRT, and as the ADRT-MP molecule incorporated into DNA were examined and compared to their effects on human DNA polymerases alpha and beta. Inhibition of HIV-RT by ADRT-TP is competitive against TTP and is more potent against RNA to DNA synthesis (Ki = 0.009 microM versus Km = 3.3 microM for TTP) than it is against DNA to DNA synthesis (Ki = 0.95 microM versus Km = 16.3 microM for TTP). ADRT-TP is also a more potent inhibitor for primer elongation on RNA template than on DNA template. ADRT-TP is a poor inhibitor of human DNA polymerases alpha (Ki = 62.5 microM) and beta (Ki = 150 microM) (Chen et al., 1992). The consequences of ADRT incorporation into DNA are strikingly different for the HIV-RT and for human DNA polymerases alpha and beta. DNA polymerases alpha and beta incorporate a single ADRT-MP molecule into nascent DNA at a very slow rate and continue to elongate. They are unable to incorporate a second consecutive ADRT-MP. However, HIV-RT is able to efficiently incorporate two consecutive ADRT molecules. Incorporation of two consecutive ADRT-MP molecules by HIV-RT prevents further DNA chain elongation. Incorporation of two ADRT-MP molecules separated by one deoxyribonucleoside monophosphate (dAMP, dCMP, or dGMP) also abolishes DNA chain elongation by HIV-RT.