T cells can recognize small molecular compounds like drugs. It is thought that covalent binding to MHC bound peptides is required for such a hapten stimulation. Sulfamethoxazole, like most drugs, is not chemically reactive per se, but is thought to gain the ability to covalently bind to proteins after intracellular drug metabolism. The purpose of this study was to investigate how sulfamethoxazole is presented in an immunogenic form to sulfamethoxazole-specific T cell clones. The stimulation of four CD4(+) and two CD8(+) sulfamethoxazole-specific T cell clones by different antigen-presenting cells (APC) was measured both by proliferation and cytolytic assays. The MHC restriction was evaluated, first, by inhibition using anti-class I and anti-class II mAb, and second, by the degree of sulfamethoxazole-induced stimulation by partially matched APC. Fixation of APC was performed with glutaraldehyde 0.05%. The clones were specific for sulfamethoxazole without cross-reaction to other sulfonamides. The continuous presence of sulfamethoxazole was required during the assay period since pulsing of the APC was not sufficient to induce proliferation or cytotoxicity. Stimulation of clones required the addition of MHC compatible APC. The APC could be fixed without impairing their ability to present sulfamethoxazole. Sulfamethoxazole can be presented in an unstable, but MHC-restricted fashion, which is independent of processing. These features are best explained by a direct, noncovalent binding of sulfamethoxazole to the MHC-peptide complex.