AbstractThe aim of this study was to assess the stereoselectivity of two tebuconazole [(RS)‐1‐p‐chlorophenyl‐4,4‐dimethyl‐3‐(1H‐1,2,4‐triazol‐1‐ylmethyl)pentan‐3‐ol] enantiomers in in vitro system (rat liver microsomes). The analytes were extracted with acetic ether and concentrations were determined by high performance liquid chromatography (HPLC) with a cellulose tris(3,5‐dimethylphenylcarbamate)‐based chiral stationary phase. The degradation of rac‐tebuconazole (15 μM) followed first‐order kinetics, and the degradation of the S‐tebuconazole (t1/2 = 22.31 min) was faster than that of the R‐tebuconazole (t1/2 = 48.76 min), but no significant difference between the enantiomers was found in the respective incubation (7.5 μM for each). Kinetic assays showed that the Km was different between the two enantiomers (KmR = 14.83 ± 2.19, KmS = 12.23 ± 2.72). The interaction results revealed that there was competitive inhibition between S‐ and R‐form, and there was a significant difference between the IC50 of R‐ to S‐tebuconazole and S‐ to R‐tebuconazole (IC50R/S/IC50S/R = 4.98). Chirality. © 2011 Wiley Periodicals, Inc.