A series of 4′-ethynyl (4′-E) nucleoside analogs were designed, synthesized, and identified as being active against a wide spectrum of human immunodeficiency viruses (HIV), including a variety of laboratory strains of HIV-1, HIV-2, and primary clinical HIV-1 isolates. Among such analogs examined, 4′-E-2′-deoxycytidine (4′-E-dC), 4′-E-2′-deoxyadenosine (4′-E-dA), 4′-E-2′-deoxyribofuranosyl-2,6-diaminopurine, and 4′-E-2′-deoxyguanosine were the most potent and blocked HIV-1 replication with 50% effective concentrations ranging from 0.0003 to 0.01 μM in vitro with favorable cellular toxicity profiles (selectivity indices ranging 458 to 2,600). These 4′-E analogs also suppressed replication of various drug-resistant HIV-1 clones, including HIV-1M41L/T215Y, HIV-1K65R, HIV-1L74V, HIV-1M41L/T69S-S-G/T215Y, and HIV-1A62V/V75I/F77L/F116Y/Q151M. Moreover, these analogs inhibited the replication of multidrug-resistant clinical HIV-1 strains carrying a variety of drug resistance-related amino acid substitutions isolated from HIV-1-infected individuals for whom 10 or 11 different anti-HIV-1 agents had failed. The 4′-E analogs also blocked the replication of a non-nucleoside reverse transcriptase inhibitor-resistant clone, HIV-1Y181C, and showed an HIV-1 inhibition profile similar to that of zidovudine in time-of-drug-addition assays. The antiviral activity of 4′-E-thymidine and 4′-E-dC was blocked by the addition of thymidine and 2′-deoxycytidine, respectively, while that of 4′-E-dA was not affected by 2′-deoxyadenosine, similar to the antiviral activity reversion feature of 2′,3′-dideoxynucleosides, strongly suggesting that 4′-Eanalogs belong to the family of nucleoside reverse transcriptase inhibitors. Further development of 4′-E analogs as potential therapeutics for infection with multidrug-resistant HIV-1 is warranted.