Constructions of a transfer vector and a recombinant baculovirus using the thymidine kinase gene of the Herpes simplex virus type 1 strain F (HSV -1) were carried out. Newly cloned transfer vector, pHcgXIIIB, was constructed by insertion of the glycoprotein gX gene signal peptide sequence of Pseudorabies virus into the baculovirus vector pHcEV-IV. The gX sequence was inserted just downstream from the promoter for the polyhedrin gene of the Hyphantria cunea nuclear polyhedrosis virus (HcNPV). HSV-1 thymidine kinase(tk) gene (1.131 kb) was used as a candidate gene for transferring into the baculovirus expression system. The tk gene was inserted into a BamHI site downstream from the gX sequence-promoter for the polyhedrin gene in the pHcgXIIIB transfer vector and was transferred into the infectious lacZ-HcNPV expression vector. Recombinant virus was isolated and was named gX-TK-HcNPV. The recombinant virus produced a 45 kDa gX-TK fusion protein in Spodoptera frugiperda cells, which was confirmed by Western blot analysis. Microscopic examination of gX-TK-HcNPV-infected cells revealed normal multiplication. Fluorescent antibody staining indicated that the gX-TK fusion protein was present in the cytoplasm. These results indicated that the transfer vector successfully transferred the gX-tk gene into the baculovirus expression system.