The gene encoding the complete glycoprotein of pseudorabies virus (PRV, Yamagata-S81 strain glycoprotein gIII) has been inserted into the baculovirus transfer vector pAcYM1S derived from the nuclear polyhedrosis virus of Autographa californica (AcNPV). A Spodoptera frugiperda cell line, SF21AE, was efficiently co-transfected with the transfer vector containing the gIII gene and AcNPV DNA by cationic liposomes (Lipofectin). The gene was placed under the control of the AcNPV polyhedrin promoter and expressed to high levels by the derived recombinant virus using SF21AE. Three polypeptides of different molecular weight were expressed. The principal products were glycosylated and transported to the cell surface. The smallest product was not glycosylated. Despite their lower molecular weight, it has been established that the antigenic properties of the peptides were conserved by comparison with those of the authentic glycoprotein gIII of PRV. Immunogenicity of the expressed products was also demonstrated. Intraperitoneal injection of expressed gIII induced neutralizing antibodies in mice. The results have raised the possibility that the protein expressed by baculovirus recombinant may be used to analyze biologically functional sites, develop a subunit vaccine and diagnostic antigens.