2',3'-Didehydro-3'-deoxy-4'-ethynylthymidine (4'-Ed4T), a novel thymidine analog, has more potent anti-human immunodeficiency virus type 1 (HIV-1) activity than its progenitor, stavudine (d4T). The profile of the intracellular metabolites of 4'-Ed4T was qualitatively similar to that of zidovudine (AZT) but not to that of d4T, while after drug removal it showed more persistent anti-HIV activity than AZT or d4T in cell culture. When CEM cells were exposed to various concentrations of 4'-Ed4T, 4'-Ed4T was efficiently taken up by the cells and was readily phosphorylated to 4'-Ed4T monophosphate (4'-Ed4TMP), 4'-Ed4T diphosphate (4'-Ed4TDP), and 4'-Ed4T triphosphate (4'-Ed4TTP). Most importantly, 4'-Ed4TTP, the active metabolite of 4'-Ed4T, persisted significantly longer than 4'-Ed4TDP and 4'-Ed4TMP after drug removal. We further investigated the efflux profiles of 4'-Ed4T in the comparison with those of AZT in CEM cells. After drug removal, both 4'-Ed4T and AZT were effluxed from the cells in a time- and temperature-dependent manner. However, the efflux of 4'-Ed4T from cells was much less efficient than that of AZT. 4'-Ed4T was effluxed from cells only in its nucleoside form, while AZT was effluxed from cells in both its nucleoside and monophosphate forms. The mechanism-of-action study showed that the efflux of 4'-Ed4T or AZT nucleoside might be due to unknown nucleoside transporters which were not related to the equilibrative nucleoside transporters, while the efflux of AZT monophosphate might be due to multidrug resistance protein 4 (MRP4/ABCC4). The results demonstrated that no detectable 4'-Ed4TMP efflux and the less efficient efflux of 4'-Ed4T nucleoside from cells might be one of the biochemical determinants of its persistent antiviral activity in cell culture.