Here we describe generation of Aw3.18, a monoclonal antibody that recognizes peptide residues 48-62 of hen egg lysozyme (HEL) bound to the MHC class II molecule I-Ak. Epitope mapping revealed that Aw3.18 detects a change in the solvent-exposed surface of this peptide-MHC complex upon substitution of the peptide side chain at position P1. Furthermore, Aw3.18 blocked recognition by some, but not all, of the HEL 48-62-reactive T cell hybridomas tested, suggesting a heterogeneity in the T cell response toward this complex. Finally, using Aw3.18, it was possible to determine the fraction of I-Ak molecules loaded with 48-62 peptide after culture of an antigen-presenting cell in medium containing HEL.