Functional analysis of mutant class II major histocompatibility complex molecules has begun to identify regions important for antibody binding and for T-cell activation. By using in vitro mutagenesis directed at the β1 domain of the Ab(β) gene we have constructed three structurally distinct mutant Ab(β) genes. Each of these genes, as well as the wild-type Ab(β) gene, was cotransfected together with the wild-type Ab(α) gene into the Ia-negative B-lymphoma cell line M12.C3. Transfection resulted in the successful synthesis and cell surface expression of three mutant class II antigens that showed serological and functional alterations as compared to the I-Ab antigens from the M12.C3 cell transfected with the wild-type gene. The variable patterns of both I-Ab-specific monoclonal antibody binding and activation I-Ab-specific T-cell hybridomas show that the mutations result in the loss of structural epitopes required for both monoclonal antibody binding and for T-cell recognition. The data suggest that there are multiple sites on a single Ia molecule that are recognized by T helper cells and also that the tertiary conformation of the Ia molecule can be critical in the formation of such sites.