1. The 4-hydroxylation of propranolol by rat and human liver microsomes is associated with formation of a chemically reactive species which binds irreversibly to cytochrome P4502D6 (CYP2D6) destroying its catalytic function. Therefore, the effect of propranolol treatment (80 mg twice daily) on debrisoquine phenotype was examined, to see if it resulted in phenocopying in vivo. The role of 4-hydroxypropranolol (4OHP) in the inhibition of CYP2D6 activity was also studied using microsomes from yeast expressing CYP2D6 and from human livers; metoprolol was used as the CYP2D6 substrate. 2. Although a significant effect on apparent oxidation phenotype was demonstrated, the absolute change in the urinary debrisoquine/4-hydroxydebrisoquine ratio (D/4HD) was small, such that no extensive metaboliser who received propranolol treatment was reclassified as a poor metaboliser. The in vitro studies indicated that 4OHP is a potent inhibitor of metoprolol metabolism (Ki approximately 1 microM). This inhibitory effect was enhanced when 4OHP was pre-incubated in the presence of a NADPH generating system and human liver microsomes. The effect was decreased significantly when reduced glutathione was added to the pre-incubation mixture. Metabolism of 4OHP occurred when incubated with human liver microsomes in the presence of a NADPH generating system and irrespective of CYP2D6 phenotype; yeast expressing CYP2D6 did not metabolise 4OHP. 3. We conclude that, although treatment with propranolol 80 mg twice daily significantly decreases the catalytic function of CYP2D6, the inhibition is insufficient to result in phenocopying. The reactive intermediate produced by further metabolism of 4OHP is probably scavenged effectively in vivo by glutathione and other nucleophiles.