AbstractAn improved procedure is described for polypeptide analysis of radiolabeled antigens resolved by crossed immunoelectrophoresis (CIE). The method involves detection of immunoprecipitates by autoradiography of CIE gels dried onto filter paper. This modification allows selected segments of immunoprecipitate arcs to be excised with a high degree of precision. Radiolabeled antigens are extracted from excised precipitates by incubation at 60 °C in Laemmli sample buffer, and polypeptides are visualized by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis performed in conjunction with autoradiography or fluorography. Protein antigens of the bacterial outer membrane are shown to retain (in part) their properties of heat‐modifiability following drying and extraction, thus facilitating their identification. The procedure is applied to the analysis of fourteen membrane‐associated antigens ofEscherichia coliand results in the identification of theompF/Cand theompAgene products, and the resolution of two novel heterooligomeric outer membrane protein antigens. The polypeptide composition of four previously uncharacterized inner membrane antigens is also established. In addition, six antigens which had been characterized by other unrelated methods as common protein antigen, β‐galactosidase, adenosine‐5′‐triphosphatase, dihydrolipoyl dehydrogenase, D‐lactate dehydrogenase and thelppgene product (the Braun lipoprotein) are shown to possess polypeptide profiles which confirm the initial identification. Evidence is also presented to support the thesis that the bound form of thelppgene product can associate with other proteins of t