Characterization of glutathione conjugation in vivo was performed in 12 healthy male volunteers by use of the racemic drug bromisovalum (bromisoval; 2‐bromoisovalerylurea) as a model substrate. To study whether the pharmacokinetics of both bromisovalum enantiomers was related to the glutathioneS‐transferase class Mu phenotype, six subjects who were class Mu deficient and six subjects who were not class Mu deficient participated. After oral administration of 600 mg racemic bromisovalum, enantioselective measurement of unchanged bromisovalum (plasma and saliva) and the diastereomeric bromisovalum mercapturates (urine) showed a pronounced stereoselectivity in all subjects. The plasma clearance ofR‐bromisovalum was about 12 times higher than that ofS‐bromisovalum (9.3 ± 3.7 and 0.78 ± 0.38 L/min, respectively), which was in agreement with the higher urinary cumulative excretion for the mercapturate derived fromR‐bromisovalum: 26% ± 4% of the dose versus 8% ± 3% of the dose for the mercapturate derived fromS‐bromisovalum. Both the bromisovalum pharmacokinetics in general and the stereoselectivity in bromisovalum pharmacokinetics were not different for the subjects who were glutathioneS‐transferase class Mu deficient and the subjects who were not glutathione transferase class Mu deficient.Clinical Pharmacology and Therapeutics(1993)53,49–58; doi