The aim of this study was to characterize the epitope specificity and the idiotype(s) of gp330-specific autoantibodies in active Heymann nephritis (HN). To study this, two monoclonal autoantibodies (mAb), 3D9B and 5B8C, derived from Lewis rats with HN, were generated. These antibodies bound to different epitopes on the autoantigen gp330, as shown by in vitro studies. In order to characterize the idiotype(s) of the mAb, rabbit anti-idiotypic antisera were prepared. It was shown that the rabbit antisera directed against the 3D9B as well as the 5B8C idiotype were able to inhibit the binding of nephritic eluate to gp330. This indicated that the idiotypes recognized on 3D9B and 5B8C were relevant to the spectrum of idiotypes involved in HN. In order to investigate the nature of the two idiotypes of these mAb, cross-absorption and binding studies were performed. The rabbit antisera raised against one of the two idiotypes bound in a direct binding ELISA to both idiotypes. Since the observed cross-reactivity of the antisera suggested that the idiotypes of 3D9B and 5B8C were at least partly cross-reactive, homologous polyclonal and monoclonal anti-idiotypic antibodies were prepared by immunizing rats with 3D9B or 5B8C. The results from the binding and inhibition experiments strongly suggest that the idiotypes of 3D9B and 5B8C contain cross-reactive or public idiotopes.