The reverse transcriptase inhibitor, nevirapine (NVP), causes skin rashes and hepatotoxicity. We used a rat model to determine if the rash is caused by the parent drug or a reactive metabolite. By manipulation of metabolic pathways and testing analogues, we eliminated all but one pathway, 12-hydroxylation, which involves the oxidation of an exocyclic methyl group, as being responsible for the rash. Treatment with 12-OH-NVP caused a rash, and an analogue in which the methyl hydrogens were replaced by deuterium to inhibit the 12-OH pathway did not cause a rash; however, quite unexpectedly, blood levels of the deuterated analogue were very low. This is due to partitioning of the benzylic free radial intermediate between oxygen rebound to form 12-OH-NVP and loss of another hydrogen atom to form a reactive quinone methide, which inactivates P450. Cotreatment with the P450 inhibitor, 1-aminobenzotriazole, led to comparable levels of NVP and the deuterated analogue, and the deuterated analogue still caused a lower rash incidence. These data clearly point to the 12-hydroxy pathway being responsible for NVP skin rash. We propose that the hepatotoxicity of NVP in humans is due to the quinone methide formed by P450 in the liver, while the skin rash may be due to the quinone methide formed in the skin by sulfation of 12-OH metabolite followed by loss of sulfate. This is the first example in which a valid animal model of an idiosyncratic drug reaction was used to determine the metabolic pathway responsible for the reaction.