Metabolism of halazepam [7-chloro-1,3-dihydro-5-phenyl-l-(2,2,2-trifluoroethyl)-W-l,4-benzodiazepin-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 S 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 3hydroxy-HZ (3-OH-HZ) > N-desalkylhalazepam (NDZ, also known as Ndesmethyldiazepam and nordiazepam) S 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 Fm). 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 3W3S enantiomer ratios of 37/63 (untreated), 55/45 (PB-treated), and 36/64 (3MC-treated1, respectively. N-dealkylation of 3-OH-HZ by liver microsomes from PB-treated rats was substrate enantioselective; the 3R-enantiomer was N-dealkylated faster than 3s-enantiomer. The results indicated that the stereoselective C3-hydroxylation of HZ is dependent on the cytochromes P-450 present in the rat liver microsomal preparations; pro-R in liver microsomes from PB-treated rats and pro-S in liver microsomes from untreated and 3MC-treated rats, respectively.