Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) glycoprotein G (gG-1 and gG-2) were expressed in insect cells from recombinant baculoviruses (AcDSMgG-1 and AcDSMgG-2, respectively) constructed using a novel baculovirus transfer vector, pAcDSM. This vector allows the coding region of a foreign gene to be precisely linked to the baculovirus polyhedrin gene at the translation initiation site and retains the native polyhedrin translation initiation environment. Fourfold more gG-1, with a higher ratio of glycosylated to unglycosylated product, was produced by AcDSMgG-1 than by Ac373'gG-1, a recombinant baculovirus which differs from AcDSMgG-1 by the presence of 21 extraneous nucleotides in the 5' nontranslated sequence. gG-1 and gG-2 expressed in recombinant baculovirus-infected insect cells undergo cotranslational N-linked glycosylation, but the overall processing of the proteins differs from that observed in HSV-1 or HSV-2-infected cells. Despite these differences, baculovirus-expressed gG-1 and gG-2 were recognized in a HSV type-specific manner by human serum specimens.