Although protein-derived nominal Ags have, in many instances, been precisely determined, the epitopes recognized by hapten-specific CD4+ T cells responsible for contact sensitization have not been defined. To better understand the nature of the precise epitopes generated after hapten interaction with Langerhans cells (LC), we assessed the ability of TNP-modified I-Ak- and I-Au-binding peptides to activate hapten-specific CD4+ T cells obtained respectively from TNCB-primed C3H (H-2k) and PL/j (H-2u) mice. Using LC as APC, I-Ak-restricted TNP-specific CD4+ T cells proliferated in the presence of the synthetic peptide hen egg lysozyme 52-61 derivatized with TNP at position 56, and less so when TNP was coupled at positions 53 or 59. Similarly, I-Au-restricted TNP-specific CD4+ T cells from PL/j mice were triggered by the synthetic I-Au-binding 13 mer poly(A)-Y5-R6 TNP-modified at position 4, and to a limited extent with TNP coupled in positions 7 or 10. Our results indicate that hapten-modified MHC class II binding nonautologous peptides are recognized by hapten-specific CD4+ T cells and that precise positioning of hapten molecules on peptides binding MHC class II molecules is required for optimal CD4+ T cell recognition. These findings provide insight into the manner in which haptens are recognized by T cells involved in contact sensitivity and should facilitate the study and design of specific therapies for the manipulation of hapten-specific CD4+ T cell responses.