To investigate the role that isotypic residues play during interactions between CD4+ T cells and MHC class II molecules, interisotypic MHC class II beta-chains have been generated in which the beta 1 domain is derived from I-A and the beta 2, transmembrane, and intracytoplasmic regions are derived from I-E. Interisotypic or wild-type A beta genes have been transfected into L cells with the genes encoding the wild-type A alpha-chain. Transfectants bearing the recombinant beta-chain thus express an MHC class II dimer in which the amino-terminal domains that control TCR and peptide-binding interactions are wild-type A alpha and A beta, whereas the membrane proximal domains are derived from A alpha and E beta. L cells expressing this recombinant class II molecule or wild-type A alpha A beta have been compared functionally for their ability to stimulate Ag-specific T cell hybridomas and normal T cell clones and to activate primary alloreactive and superantigen-specific T cells. Ag-specific T cell hybridomas vary in their ability to be activated by the recombinant class II molecule, and sensitivity to the isotypic form of the membrane proximal domain correlates with expression of the CD4 molecule. CD4+ T cells distinguish between the wild-type and recombinant dimers, whereas CD4- T cells react equivalently with both. The recombinant class II molecules are defective in activation of normal T cell clones and are totally deficient in activation of primary alloreactive and superantigen-reactive T cells, but stimulate CD4- alloreactive T cell hybridomas equivalently. Together, the results from these experiments suggest that the interisotypic dimers possess normal TCR and peptide interactions, but altered CD4-dependent accessory interactions necessary for activation of normal T cells. These findings indicate that isotypic residues in the membrane proximal domains of MHC class II control CD4-linked accessory function and that this accessory function is most critical for freshly isolated CD4 lymphocytes.