The drug procainamide (PA) is notorious for causing drug-induced systemic lupus erythematosus (SLE) in humans. Indirect evidence suggests that metabolism of PA to a reactive intermediate metabolite is involved in the pathogenesis of drug-induced SLE in that N-hydroxylation of the arylamine group of PA favors this condition, whereas N-acetylation prevents it. If this is correct, one would expect hydroxylamine-PA (HAPA) to be immunogenic, whereas N-acetyl-PA (N-ac-PA) should be nonimmunogenic. This hypothesis was confirmed by means of the popliteal lymph node assay (PLNA) in mice: injection of PA and N-ac-PA failed to induce a reaction in the direct PLNA, whereas HAPA induced a vigorous reaction. Using the adoptive transfer PLNA, splenic T cells of mice that had received three injections of HAPA were shown to be specifically sensitized to this metabolite, but not to PA or N-ac-PA. In this system, an anamnestic T cell response could also be elicited when homogenized peritoneal cells of mice that had been treated with PA for 4 months were used as the challenging antigen, indicating that the peritoneal cells of PA-treated animals contained or had been exposed to the reactive intermediate metabolite HAPA. Whereas in slow acetylator mice this 4-month PA treatment sufficed to generate HAPA in peritoneal cells, fast acetylators required additional stimulation of their oxidative metabolism in order to produce enough HAPA detectable by sensitized T cells. These findings clearly support the concept that reactive intermediate metabolites, such as HAPA, are generated by the oxidative metabolism of phagocytic cells and are immunogenic for T cells.