The elucidation of the structural basis for expression of the cross-reactive Id of the A strain mouse (CRIA) in response to the hapten p-azophenylarsonate has been the object of considerable research effort. Most conclusions regarding the amino acids involved in Id expression have been inferential, based on comparisons of amino acid sequences, chain recombination experiments, or chemical modification of particular amino acids. To more rigorously designate the amino acids critical to the phenotype of the CRIA, a system for the expression and directed mutation of antibody molecules was developed. Based on the baculovirus expression of foreign proteins in cultured insect cells, functional antibodies can be produced at very high levels in vitro. By the process of oligonucleotide-directed mutagenesis, a series of specific amino acid changes were introduced into both the H and L chains of a prototype CRIA expressing antibody molecule. Analysis of the gain or loss of polyclonal Id and each of several monoclonal idiotopes has allowed us to identify more precisely the amino acids responsible for expression of the CRIA. Thus we have shown that expression of the CRIA is equally dependent on amino acids in the second and third CDR of the H chain. Furthermore, the idiotopes studied surround the Ag-binding site and clearly involve multiple interactions in several of the L and H chain CDR.