AbstractMetabolism of halazepam [7‐chloro‐1,3‐dihydro‐5‐phenyl‐1‐(2,2,2‐trifluoroethyl)‐2H‐1,4‐benzodi epin‐2‐one, HZ] was studied by incubation with liver microsomes prepared from untreated, phenobarbital (PB)‐treated, and 3‐methylcholanthrene (3MC)‐treated male Sprague–Dawley rats. Metabolites of HZ were separated by normal‐phase HPLC. Relative rates of HZ metabolism by liver microsomes prepared from untreated and treated rats were PB‐treated ≫ untreated>3MC‐treated at low concentration of microsomal enzymes (0.25 mg protein per ml of incubation mixture) and PB‐treated ≫ 3MC‐treated ≈ untreated at high concentration of microsomal enzymes (2 mg protein per ml of incubation mixture). The relative amounts of major metabolites were found to be 3‐hydroxy‐HZ (3‐OH‐HZ)>N‐desalkylhalazepam (NDZ, also known asN‐desmethyldiazepam and nordiazepam) ≫ oxazepam (OX) for all three rat liver microsomal preparations and the distribution of metabolites was independent of microsomal enzyme concentrations. Enantiomers of 3‐OH‐HZ were resolved by HPLC on a Chiralcel OC column (cellulose trisphenylcarbamate coated on silica gel, particle size 10 μm). 3‐OH‐HZ enantiomeres have racemization half‐lives of ∼ 150 min in pH 4,7.5, and 10 aqueous solutions. 3‐OH‐HZ formed in the metabolism of HZ by liver microsomes prepared from untreated and treated rats were found to have 3R/3S enantiomer rations of 37/63 (untreated), 55/45 (PB‐treated), and 36/64 (3MC‐treated), respectively.N‐dealkylation of 3‐OH‐HZ by liver microsomes from PB‐treated rats was substrate enantioselective; the 3R‐enantiomer wasN‐dealkylated faster than 3S‐enantiomer. The results indicated that the stereoselective C3‐hydroxylation of HZ is dependent on the cytochromesP‐450 present in the rat liver microsomal preparations; pro‐R in liver microso