ABSTRACT 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-1 _M41L/T215Y , HIV-1 _K65R , HIV-1 _L74V , HIV-1 _{M41L/T69S-S-G/T215Y} , and HIV-1 _{A62V/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-1 _Y181C , 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′- E analogs 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.