(S)-1-[3-hydroxy-2-(phosphonomethoxy)propyl]cytosine (HPMPC [cidofovir]) and (S)-9-[3-hydroxy-2-(phosphonomethoxy)propyl]adenine (HPMPA) are potent inhibitors of a variety of DNA viruses. These drugs possess a 3'-hydroxyl equivalent which could support chain extension from an incorporated drug molecule. HPMPC and HPMPA were initially reported to lack activity against human immunodeficiency virus type 1 (HIV-1); more recent results have shown that the octadecyloxyethyl (ODE) and hexadecyloxypropyl (HDP) esters of HPMPA are potent inhibitors of the virus. We have synthesized the ODE esters of a series of (S)-[3-hydroxy-2-(phosphonomethoxy)propyl] (HPMP) nucleosides, including HPMPC, HPMP-guanine (HPMPG), HPMP-thymine (HPMPT), and HPMP-diaminopurine (HPMPDAP), as well as the ODE ester of the obligate chain terminator (S)-9-[3-methoxy-2-(phosphonomethoxy)-propyl]adenine (MPMPA). All compounds except ODE-HPMPT were inhibitors of HIV-1 replication at low nanomolar concentrations. These compounds were also inhibitors of the replication of HIV-1 variants that are resistant to various nucleoside reverse transcriptase (RT) inhibitors at concentrations several times lower than would be expected to be achieved in vivo. To investigate the mechanism of the antiviral activity, the active metabolites of HPMPC and HPMPA were studied for their effects on reactions catalyzed by HIV-1 RT. Incorporation of HPMPC and HPMPA into a DNA primer strand resulted in multiple inhibitory effects exerted on the enzyme and showed that neither compound acts as an absolute chain terminator. Further, inhibition of HIV-1 RT also occurred when these drugs were located in the template strand. These results indicate that HPMPC and HPMPA inhibit HIV-1 by a complex mechanism and suggest that this class of drugs has a broader spectrum of activity than previously shown.