Class II major histocompatibility complex heterodimers present to T cells determinants as sets of antigen fragments with ragged N and C termini. It is not yet elucidated whether different types of antigen-presenting cells generate identical sets of peptides containing the same determinant. Taking advantage of recombinant I-Ed molecules produced by insect cells as empty heterodimers, a sensitive T cell stimulation assay was developed to analyze naturally processed hen egg lysozyme (HEL) peptides. I-Ed preparations were isolated from antigen-presenting cells cultured with HEL. Following acid treatment, peptides eluted from I-Ed were chromatographed and the fractions incubated at acidic pH with purified recombinant I-Ed molecules, conditions which favor peptide binding. The stimulatory capacity of the reconstituted peptide-I-Ed complexes adsorbed on the well surface of cell culture plates was then evaluated by measuring interleukin-2 secreted by an HEL 107-116-specific, I-Ed-restricted T cell hybridoma. We found that the B lymphoma A20 and an I-Ed-transfected fibroblast cell line generated distinct sets of peptides containing the HEL sequence 107-116. Our results suggest the possibility that presentation of one determinant by different types of antigen-presenting cells stimulates populations of T cells with distinct fine antigen specificities.