Reverse transcriptase (RT) is an essential enzyme for the replication of HIV and thus is regarded as one of the most important targets for anti-HIV agents. So far, only nucleoside RT inhibitors such as 3'-azido-3'-deoxythymidine (AZT), 2',3'-dideoxyinosine (ddI), 2',3'-dideoxycytidine (ddC), and 2',3'-didehydro-3'-deoxythymidine (d4T) have been approved for the treatment of HIV-infected patients, but their long-term use leads to toxic side effects like bone marrow suppression. Non-nucleoside inhibitors such as 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine, dipyridodiazepinone (Nevirapine), and α-anilinophenylacetamide (R-89439, Loviride) have been developed as specific and potent inhibitors of HIV-1 RT. From a random screening program of compounds with herbicidal activities, we identified a series of 1,2,5-thiadiazoles (TDA) as inhibitors of HIV-1. In this communication, we describe the efficient synthesis, anti-HIV-1 activity, and structure-activity relationship of novel non-nucleoside inhibitors with the thiadiazole skeleton. The 3-hydroxyl-4-substituted-1,2,5-thiadiazole nucleus was constructed in good yield by condensing α-amino acid amides with sulfur monochloride in dimethylformamide. The carbamoyl moiety was site-selectively introduced at the 3-hydroxy position of the thiadiazole skeleton using two methods: treatment with carbamoyl chlorides or reaction with triphosgene followed by secondary amines. Antiviral activity was assessed based on inhibition of HIV-1-induced cytopathic effect (CPE) in MT-4 cells or reduction of HIV-1 p24 antigen in peripheral blood lymphocytes (PBLs). The 4-(2,6-dichlorophenyl)-1,2,5-thiadiazol-3-yl N-methyl-N-propylcarbamate (19) exhibited the most potent activity, with an EC50 of 0.013 μM and a CC50 of 131 μM in MT-4 cells (selectivity index = 10 077) and an EC50 of 0.004 μM in PBLs, while it had no effect on HIV-2 replication at concentrations up to 131 μM. Structure-activity relationship studies indicated that the presence of both an aromatic ring at the C-4 position and a carbamate moiety at the C-3 position of the thiadiazole ring was essential for activity. In conclusion, TDA derivatives are highly potent and specific inhibitors of HIV-1. Considering their ease of synthesis, they have potential as candidate drugs for HIV-1 chemotherapy. Mechanistic studies revealed they belong to the family of nonnucleoside HIV-1 RT inhibitors, and further investigations on structure-activity relationships and drug resistance emergence are in progress.