The binding sites of class II major histocompatibility complex (MHC) molecules can accommodate many seemingly diverse peptides. In the case of mouse class II molecules, it appears that in general, the I-A and I-E isotypes associate with different peptides. In this study we report an example where a single amino acid substitution in an I-Ak restricted peptide changes the restriction element to I-Ek. A T cell hybridoma, F6.A10, specific for the peptide 93-104 from mouse testicular cytochrome c (Mt cyt 93-104) was found to be restricted by I-Ak using class II molecule specific blocking monoclonal antibodies (mAb). The activation of this hybridoma by Mt cyt 93-104 was competitively inhibited by other peptides that bind to the I-Ak molecule but not by the peptide Mt cyt 93-104(A96) in which lysine at position 96 was substituted by alanine. This single amino acid substitution resulted in the ability of Mt cyt 93-104(A96) to activate the pigeon cytochrome c specific, I-Ek restricted, T cell hybridoma 2B4.11. The activation of 2B4.11 by Mt cyt 93-104(A96) was inhibited by peptides which bind to the I-Ek molecule but not by Mt cyt 93-104 and by mAb specific for I-Ek but not by mAb specific for I-Ak. These results suggest that the amino acid at position 96 may be an important anchor residue for both I-Ak and I-Ek binding but that peptides with different amino acid side chains are accommodated at that position by one or the other MHC class II isotype. Thus, in this particular case a single amino acid residue in the peptides determines the MHC class II isotype specificity.