Nevirapine, used for the treatment of HIV infection, is associated with development of skin rash and liver toxicity. The mechanism of these idiosyncratic reactions is unknown. We have previously reported the discovery of a new animal model of nevirapine-induced skin rash in rats. When treated with nevirapine, Brown Norway rats developed red ears on about day 7 and skin rash on about day 21. On rechallenge, ears turn red within 24 h, and skin lesions develop by day 9. In the current study, we analyzed the time course of the sequence of events involved in the development of skin rash. Rats were treated with nevirapine for 7, 14, or 21 days or rechallenged with it for 0, 1, or 9 days. This treatment led to an increase in the total number of auricular lymph node T, B, and macrophage cells. There was also an increase in the activation/infiltration marker ICAM-1 and activation/antigen presentation marker MHC II in these cells compared with those from control rats. Immunohistochemistry analysis showed macrophage infiltration and ICAM-1 expression in the ears of treated rats as early as day 7 of treatment. Macrophage infiltration preceded T cell infiltration, which was not apparent until the onset of rash. Both MHC I and MHC II expression increased in the skin of nevirapine-treated rats that developed rash. A major inducer of MHC is IFNgamma. Although rechallenge with nevirapine led to a large increase in serum levels of IFNgamma, this was not observed during the treatment of naïve rats with nevirapine. These observations provide further clues to the mechanism of nevirapine-induced skin rash.