AbstractStereoselective metabolism of propranolol side‐chain glucuronidation was studied for two recombinant human uridine diphosphate glucuronosyltransferases (UGTs), UGT1A9 and UGT2B7. TheS‐ andR‐propranolol side‐chain glucuronides produced in the incubation mixtures were assayed simultaneously by RP‐HPLC with fluorescent detector. The excitation and emission wavelengths were set at 310 nm and 339 nm, respectively. UGT1A9 prefers catalyzingS‐enantiomer toR‐enantiomer and the intrinsic clearance (CLint) ratios ofS‐enantiomer toR‐enantiomer are 3.8 times and 6.5times for racemic propranolol and individual enantiomers, respectively. UGT2B7, however, catalyzes slightly lessS‐enantiomer thanR‐enantiomer and the CLintratio ofS‐enantiomer toR‐enantiomer is 0.8 times. The high concentration of racemic propranolol (>0.57 mmol/l) and individual enantiomers (>0.69 mmol/l) exhibited substrate inhibition of glucuronidation for UGT2B7, but only theS‐enantiomer (>0.44 mmol/l) in racemic propranolol exhibited substrate inhibition for UGT1A9. The substrate inhibition constants (Ksi) were all similar (P> 0.05). Drug–drug interactions were also found betweenS‐ andR‐enantiomer glucuronidation metabolisms by UGT1A9 and UGT2B7. Chirality 2010. © 2009 Wiley‐Liss, Inc.