In the search for novel derivatives of 1-[2-(hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT), we have found that several 5-ethyl-6-(3,5-dimethylphenylthio)uracil and 5-ethyl-6-(3,5-dimethylbenzyl)uracil analogues are exquisitely potent and selective inhibitors of human immunodeficiency virus type 1 (HIV-1) replication in a variety of cell culture systems. Of this series, 5-ethyl-1-ethoxymethyl-6-(3,5-dimethylbenzyl)uracil (E-EBU-dM) emerged as the most active congener. Its 50% inhibitory concentration for HIV-1 (HTLV-IIIB) in MT-4 cells and peripheral blood lymphocytes is 2.2 and 0.45 nM, respectively. These concentrations are more than 10(5)-fold lower than the 50% cytotoxic concentrations of E-EBU-dM for the host cells. All compounds proved equally inhibitory to a number of clinical HIV-1 isolates, including a 3'-azido-2',3'-dideoxythymidine-resistant variant. However, as previously noted for HEPT, they do not inhibit human immunodeficiency virus type 2 replication. Reverse transcriptase assays have revealed that these HEPT derivatives act specifically on HIV-1 reverse transcriptase, according to a mechanism that is different from that of the dideoxynucleosides.