The humoral response of mice to Staphylococcus-aureus nuclease was previously shown to be controlled genetically by an H-2-linked Ir [immune response] gene(s). To examine the possible contributions of variable region immunoglobulin genes to this genetic control, a system for the detection of idiotypic determinants on anti-nuclease immunoglobulin molecules was developed. Antisera to nuclease were raised in 2 high responder strains, A/J and SJL. The corresponding antibodies were purified by affinity chromatography on Sepharose-nuclease columns, and were used to immunize groups of Lewis rats. An assay system was developed to assess the inhibition of antibody-mediated inactivation of nuclease activity by the rat antisera thus produced. Despite the presence of many species-specific anti-mouse immunoglobulin antibodies in these sera, inhibition of antibody-mediated enzyme inactivation was specific for anti-nuclease antibodies of the immunizing strain. The inhibition could not be removed by extensive absorption with normal serum proteins from the antibody-producing strain, and required antibodies directed toward binding sites of the anti-nuclease antibodies. This inhibition thus defines idiotypic determinants of anti-nuclease antibodies.