Salmeterol xinafoate (Serevent) is a long-acting beta2-adrenoceptor agonist, used in the treatment of asthma, that has bronchodilator and anti-inflammatory action. Salmeterol is extensively metabolized by aliphatic oxidation in humans, with the major metabolite being alpha-hydroxysalmeterol. The aim of this investigation was to identify the specific cytochrome P450 (P450) isoform or isoforms involved in the formation of alpha-hydroxysalmeterol in human liver microsomes. [14C]Salmeterol was incubated with a pooled sample (N = 19) of human liver microsomes in the absence or presence of selective chemical inhibitors of the major human P450 isoforms. One microM ketoconazole, a selective inhibitor of CYP3A, substantially inhibited the metabolism of salmeterol to alpha-hydroxysalmeterol. Disulfiram caused a small but consistent decrease in the amount of alpha-hydroxysalmeterol formed, possibly reflecting less than total selectivity for CYP2E1 under the conditions used. Other selective inhibitors had no significant effect on the metabolism of salmeterol. The rates of formation of alpha-hydroxysalmeterol in 10 individual liver microsomal samples showed an approximately 10-fold variation and were found to be highly correlated (r2 = 0.94; p < 0.001) with rates of metabolism of midazolam to 1'-hydroxymidazolam, a marker of CYP3A activity, in the same microsomal samples. No significant correlation was evident for the metabolism of salmeterol with levels of total P450 or other markers of human P450 activities in the same microsomal samples, thus indicating that the formation of alpha-hydroxysalmeterol is catalyzed predominantly by CYP3A. Insect cell microsomes that coexpressed human CYP3A and NADPH-P450 reductase were able to metabolize [14C]salmeterol to alpha-hydroxysalmeterol, thus confirming the role of CYP3A in catalyzing this reaction. The therapeutic dose of salmeterol is very low, so it is unlikely that any clinically relevant interactions will be observed as a consequence of the coadministration of salmeterol and other pharmaceutical agents that are metabolized by CYP3A.