Chiral　compounds　usually　behave　enantioselectively　in　phyto-biochemical　processes.　Imidazolinones　are　a　class　of　chiral　herbicides　that　are　widely　used.　They　inhibit　branched-chain　amino　acid　biosynthesis　in　plants　by　targeting　acetolactate　synthase　(ALS).　It　has　been　reported　that　the　imidazolinone　enantiomers　show　different　inhibiting　activities　to　maize　(Zea　mays　L.)　seedlings　and　ALS.　However,　to　date,　the　mechanism　of　enantioselective　inhibition　of　imazethapyr　(IM)　on　ALS　activity　has　not　been　well　studied.　In　this　study,　pure　enantiomers　of　1M　were　used　for　characterizing　their　differences　in　activity　to　ALS.　Computational　molecular　docking　was　performed　to　discover　the　molecular　interaction　between　IM　enantiomers　and　ALS　at　the　first　time.　Results　showed　that　the　IM　enantiomers　enantioselectively　suppressed　the　in　vitro　and　in　vivo　ALS　activity　of　maize　leaves.　R-(-)-IM　was　more　active　than　S-(+)-IM.　The　in　vivo　ALS　activity　study　showed　only　a　2-fold　difference　between　R-(-)-IM　and　S-(+)-IM.　Quite　different　from　the　in　vivo　study,　the　in　vitro　study　showed　that　the　difference　in　inhibition　between　the　enantiomers　fell　sharply　as　concentration　increased.　At　the　lowest　concentration　of　40　mu　g　L-1,　R-(-)-IM　appeared　25　times　more　active　than　S-(+)-IM,　but　only　7　times　at　200　mu　g　L-1.　At　the　highest　concentration　of　25　mg　L-1,　in　vitro　ALS　activity　was　almost　completely　inhibited　by　S-(+)-,　R-(-)-IM　and　(+/-)-IM,　there　was　only　1.1　times　differences　between　S-(+)-　and　R-(-)-IM.　Molecular　modeling　results　provide　the　rational　structural　basis　to　understand　the　mechanism　of　enantioselective　inhibition　of　IM　on　ALS　activity.