A series of nucleosides were synthesized in which the 4'-hydrogen was substituted with either an azido or a methoxy group. The key steps in the syntheses of the 4'-azido analogues were the stereo- and regioselective addition of iodine azide to a 4'-unsaturated nucleoside precursor followed by an oxidatively assisted displacement of the 5'-iodo group. The 4'-methoxynucleosides were made via epoxidation of 4'-unsaturated nucleosides with in suit epoxide opening by methanol. Reaction-mechanism considerations, empirical conformation rules, NMR-based conformational calculations, and NOE experiments suggest that the 4'-azidonucleosides prefer a 3'-endo (N-type) conformation of the furanose moiety. When evaluated for their inhibitory effect on HIV in A3.01 cell culture, all the 4'-azido-2'-deoxy-beta-D-nucleosides exhibited potent activity. IC50's ranged from 0.80 microM for 4'-azido-2'-deoxyuridine (6c) to 0.003 microM for 4'-azido-2'-deoxyguanosine (6e). Cytotoxicity was detected at 50-1500 times the IC50's in this series. The 4'-methoxy-2'-deoxy-beta-D-nucleosides were 2-3 orders of magnitude less active and less toxic than their azido counterparts. Modifications at the 2'- or 3'-position of the 4'-substituted-2'-deoxynucleosides tended to diminish activity. Further evaluation of 4'-azidothymidine (6a) in H9, PBL, and MT-2 cells infected with HIV demonstrated a similar inhibitory profile to that of AZT. However, 4'-azidothymidine (6a) retained its activity against HIV mutants which were resistant to AZT.