The in vitro metabolism of four s-triazine herbicides (atrazine, terbuthylazine, ametryne, and terbutryne) was studied using liver microsomes from rats, pigs, and humans. New HPLC methods with UV detection were developed for the analyses of the incubations. Principal phase I reactions were N-monodealkylation, hydroxylation of the isopropyl or tert-butyl moiety, and sulfoxidation of the substrates in all species. Bidealkylation, 2-hydroxylation, or cleavage of the tert-butyl moiety could not be found in this system. The sulfoxidation of the 2-methylthio-s-triazines exceeded catalysis of the other metabolic reactions by 3-4-fold in all species. In general, all species produced the same types of metabolites, but with species-specific differences in the ratios of the metabolites. Species-specific stereoselective formation of a new chiral isopropyl-hydroxylated metabolite from atrazine was investigated using chiral HPLC techniques. The stereoselective production of this metabolite was different in the different species, with S/R ratios of 76:24 in rats, 49:51 in pigs, and 28:72 in humans.