In order to study immunological reactivity of individual enterovirus polypeptides and evaluate their usefulness for enterovirus diagnosis, the genes coding for viral structural and nonstructural proteins were expressed using recombinant baculoviruses. A histidine-tailed coxsackievirus B3 (CBV3) VP1 capsid protein was expressed and purified by immobilized metal ion affinity chromatography for EIAs. To elucidate the usefulness of the other CBV3 capsid proteins for immunoassays, recombinant baculovirus expressing the whole CBV3 capsid polyprotein region was constructed. For the detection of a potentially broader spectrum of enteroviruses, the conserved nonstructural P3 region was expressed. The P3 region encodes four nonstructural proteins including a cysteine protease (3C) and an RNA-dependent RNA-polymerase (3D). The 3C polypeptide was shown to be proteolytically active indicating that the baculovirus system is capable of expressing biologically functional enterovirus proteins. Immunoblot analysis detected antibodies against the VP1, 3C and 3D proteins in human serum samples. When the baculovirus-expressed antigens were compared with lysates of enterovirus-infected cells and a synthetic peptide in EIA highly similar results were obtained with recombinant VP1 and the lysate antigens. Although reactive in immunoblots, the P3 encoded proteins were not satisfactory for EIA.