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2′,3′-Didehydro-3′-deoxy-4′-ethynylthymidine (4′-Ed4T), is a recently discovered nucleoside reverse transcriptase inhibitor (NRTI) showing a 5- to 10-fold greater anti-human immunodeficiency virus type 1 (HIV-1) activity and less cellular and mitochondrial toxicity than its parental compound, stavudine (D4T). It is also active against a variety of NRTI-resistant HIV-1 mutants under non-cytotoxic concentrations. In this study, the effects of 4′-Ed4TTP, which is the triphosphate metabolite of 4′-Ed4T, on HIV-1 reverse transcriptase (RT) activity were investigated. We found that 4′-Ed4TTP was a substrate of HIV-1 RT serving as a DNA chain terminator, and it inhibited the DNA polymerase activity of RT more efficiently than D4TTP. The value of Ki(4′-Ed4TTP)/Km(dTTP) is 0.15 for DNA/RNA primer/template duplex (P/T), but 0.7 for DNA/DNA P/T, suggesting 4′-Ed4TTP inhibits RT more efficiently during RNA-dependent DNA synthesis than DNA-dependent DNA synthesis. 4′-Ed4TTP was also found to inhibit the 3TC (Lamivudine)-resistant RT mutant, M184V, with 3-fold less efficiency than the wild type (wt) RT. 4′-Ed4TTP showed much less inhibitory effects toward major host DNA polymerases. Overall, our results suggest that 4′-Ed4TTP is the active form for anti-HIV-1 activity via its inhibitory effect against RT.