Allergic immune responses are initiated and maintained by T cells that recognize peptidic fragments of allergens in the context of major histocompatibility complex (MHC) class II molecules. An anomaly of this model exists in the T-cell response to haptens. Haptens are nonpeptide antigens that alone are too small to provoke an immune response. Nevertheless, T-cell responses to haptenic allergens clearly occur and are critically involved in allergic immune responses to drugs such as penicillin. Although the mechanisms that generate T-cell epitopes from protein antigens are well understood, haptens create T-cell epitopes by alternative mechanisms. These may include binding of haptens directly to preformed MHC-peptide complexes on the cell surface, or indirect association with MHC molecules after conjugation with self cell surface or serum proteins that are then processed and presented as haptenated peptide antigens. Which of these unorthodox mechanisms of epitope generation is dominant in allergy to penicillin is unknown. This study aims to determine the nature of the epitopes recognized by amoxicillin-specific T cells from allergic donors, and to clarify whether T-cell responses to penicillin antibiotics are MHC-restricted and require haptenated self proteins to be processed before recognition. Human T-cell lines specific for amoxicillin were raised and used in assays with processing-disabled and MHC-class II-typed antigen-presenting cells to determine the MHC restriction and processing requirements of T cells recognizing amoxicillin. Fixation of antigen-presenting cells with paraformaldehyde, before or after pulsing with amoxicillin, established that T cells can recognize amoxicillin-containing epitopes with a similar efficiency irrespective of whether the antigenic conjugate has been internalized and processed. These results suggest that amoxicillin can bind directly to performed MHC-peptide complexes and need not necessarily involve the processing of haptenated self carrier proteins before recognition of the conjugate by amoxicillin-specific T cells.