Nevirapine treatment can cause a skin rash. We developed an animal model of this rash and determined that the 12-hydroxylation metabolic pathway is responsible for the rash, and treatment of animals with 12-OH-nevirapine also leads to a rash. In the present study, we investigated the specificity of lymphocytes in nevirapine-induced skin rash. Brown Norway rats were treated with nevirapine or 12-OH-nevirapine to induce a rash. Lymph nodes were removed, and the response of lymphocytes to nevirapine and its metabolites/analogs was determined by cytokine production (enzyme-linked immunosorbent assay, enzyme-linked immunosorbent spot assay, and Luminex) and proliferation (alamar blue assay). Subsets of lymphocytes were depleted to determine which cells were responsible for cytokine production. Lymphocytes from animals rechallenged with nevirapine proliferated to nevirapine, but not to 12-OH-nevirapine or 4-chloro-nevirapine. They also produced interferon-gamma (IFN-gamma) when exposed to nevirapine, significantly less when exposed to 4-chloro-nevirapine, and very little when exposed to 12-OH-nevirapine, even though oxidation to 12-OH-nevirapine is required to induce the rash. Moreover, the specificity of lymphocytes from 12-OH-nevirapine-treated rats was the same, i.e., responding to nevirapine more than to 12-OH-nevirapine, even though these animals had never been exposed to nevirapine. A Luminex immunoassay showed that a variety of other cytokines/chemokines were also produced by nevirapine-stimulated lymphocytes. CD4(+) cells were the major source of IFN-gamma. The specificity of lymphocytes in activation assays cannot be used to determine what initiated an immune response. This has significant implications for understanding the evolution of an immune response and the basis of the pharmacological interaction hypothesis.