Although cutaneous adverse drug reactions (ADRs) are relatively frequent and potentially severe, their mechanisms are poorly understood and no validated predictive experimental model is available. Sulfamethoxazole (SMX) is commonly used to treat infections in HIV-positive patients and severe cutaneous ADRs have been described. This study was undertaken to test whether sensitization to SMX could be achieved in mice using a combination of in vivo and in vitro endpoints. No delayed-type hypersensitivity (DTH) response could be evidenced following SMX injection in the back and subsequent challenge into the footpad or onto the ear. Pretreatment with the enzymatic inducers phenobarbitone and betanaphtoflavone, or depletion in CD4(+) T-lymphocytes were not successful either. In contrast, the injection of SMX/S9 mix in the back and challenge with SMX/S9 mix induced a significant increase in footpad thickness. A significant proliferation of spleen cells from SMX- or SMX/S9 mix-treated mice was evidenced following incubation with SMX/S9 mix, but not SMX alone. This study provides indirect evidence that SMX metabolites are involved and confirms previous in vitro results obtained with lymphocytes from patients with a history of SMX-induced ADRs cultured with murine microsomes. Further investigations using other drugs known to induce similar ADRs are warranted to establish the predictive value of this murine model.