We have studied the regio- and stereoselectivity of ring-hydroxylation and N-desisopropylation of S (-)- and R (+)-propranolol, using dog liver microsomes and the expressed dog CYP2D15 in insect cells. In dog liver microsomes, 4-hydroxylation was the preferred pathway in S (-)-propranolol oxidation, while N-desisopropylation was the preferred pathway in R (+)-propranolol oxidation. S (-)-Propranolol was preferred over R (+)-propranolol as substrate for 4- and 5-hydroxylations, while R (+)-propranolol was the preferred substrate for N-desisopropylation at higher substrate concentrations. The expressed CYP2D15 had high catalytic activities toward 4-, 5-hydroxylation, as well as N-desisopropylation of both enantiomers. At the substrate concentrations used, 4-hydroxylation was the preferred pathway for the metabolism of both enantiomers, and S (-)-propranolol was the preferred substrate over R (+)-propranolol for all three monooxygenations catalyzed by CYP2D15. Anti-CYP2D15 peptide antibody strongly inhibited 4- and 5-hydroxylation of both enantiomers in dog liver microsomes, while it did not inhibit their N-desisopropylation. These findings suggest that CYP2D15 is highly responsible for the stereoselective 4- and 5-hydroxylations of propranolol in dog liver microsomes.