4'-Acylated thymidines represent a new class of DNA chain terminators, since they have been shown to act as post-incorporation chain-terminating nucleotides despite the presence of a free 3'-hydroxyl group. Here, we describe the action of the 4'-acetyl- (MeTTP) and 4'-propanoylthymidine 5'-triphosphate (EtTTP) on HIV-1 reverse transcriptase in RNA- and DNA-dependent DNA synthesis and on DNA synthesis catalyzed by the cellular DNA polymerases alpha, beta, delta and epsilon. MeTTP exhibits a high selectivity towards HIV-1 reverse transcriptase. By the use of the bulkier propanoyl group as the 4'-substituent of the nucleoside 5'-triphosphate, selectivity towards HIV-1 reverse transcriptase could be increased without affecting substrate efficiency. Thus, 4'-modifications may serve as a tool to increase selectivity towards HIV-1 reverse transcriptase.