Thioethers are the main flavor compounds found in Liliaceae Allium vegetables and have been shown to have beneficial effects against several diseases correlated with metabolic syndrome. The inhibitory effects of six thioethers on fatty acid synthase (FAS) were investigated. Dose-dependent and time-dependent inhibitions of FAS by one trisulfide and two disulfides were revealed. Diallyl trisulfide (DATS, IC(50) = 8.37 microM) was the most active of these thioethers. Inhibition kinetics, substrate protection analysis, and stoichiometric assay revealed that DATS interacted with both essential sulfhydryl groups on the acyl-carrier protein and beta-ketoacyl synthase domain of FAS to inactivate the enzyme. The inactivation by DATS represented affinity-labeling kinetics. The active thioethers also inhibited the differentiation and lipid accumulation of 3T3-L1 preadipocytes, and the effect was related to their inhibition of FAS. It is suggested that the inhibition on FAS by thioethers and Allium vegetables is an important factor for their effects against metabolic syndrome.