Procainamide (PA) may cause drug-induced lupus, and its reactive metabolites, hydroxylamine-PA (HAPA) and nitroso-PA, are held responsible for this. Here, we show that N-oxidation of PA to these metabolites can take place in macrophages and lead to formation of neoantigens that sensitize T cells. Murine peritoneal macrophages (PMvarphi), exposed to PA in vitro, generated neoantigens related to HAPA as indicated by (1) their capacity to elicit a specific recall response of HAPA-primed T cells in the adoptive transfer popliteal lymph node (PLN) assay and (2) the appearance of metabolite-bound protein in PA-pulsed PMvarphi, as determined by Western blot. Analysis of five phase I enzymes that might be responsible for HAPA formation by PMvarphi pointed to prostaglandin H synthase-2 (PGHS-2) as a likely candidate. Experimental evidence that PA can be oxidized to HAPA by PGHS was obtained by exposing PA to PGHS in vitro. The resulting metabolites were identified by mass spectral analysis and covalent protein binding in ELISA. In vitro, PA exposure of PMvarphi of slow acetylator A/J and fast acetylator C57BL/6 mice failed to show significant strain differences in enzyme mRNA expression, enzyme activities, or formation of HAPA-related neoantigens. By contrast, after long-term PA treatment in vivo only in slow acetylators the PMvarphi harbored HAPA-related neoantigens and T cells were sensitized to them. PMvarphi of fast acetylator C57BL/6 mice only contained HAPA-related neoantigens, and their T cells were only sensitized to them if, in addition to long-term PA treatment, their donors had received injections of phorbol myristate acetate (PMA), a known enhancer of oxidative enzymes in phagocytes. In conclusion, PA treatment leads to N-oxidation of PA by enzymes, in particular PGHS-2, present in antigen-presenting cells (APC) and, hence, to generation of neoantigens which sensitize T cells. The enhanced neoantigen formation and T cell sensitization seen in slow acetylators might be explained by their higher concentration of PA substrate that is available for extrahepatic N-oxidation in APC.