The DNA polymerase gene of Epstein-Barr virus (EBV) was cloned into baculovirus transfer vector (pBlueBac). The recombinant baculovirus (AcEBP-15) was obtained by cotransfection of Spodoptera frugiperda (Sf9) cells with infectious DNA from Autographa californica multiple nuclear polyhedrin virus (AcMNPV) and pBlueBac plasmid carrying EBV polymerase gene. Infection of Sf9 cells with the recombinant virus produced substantial quantities of the EBV DNA polymerase protein of the expected size (110 kD). The identity of the EBV polymerase 110-kD polypeptide was determined by (a) immunoprecipitation and Western blot analyses with rabbit polyclonal antiserum specific for a synthetic peptide derived from the coding sequence of the polymerase gene; (b) identification of a polypeptide of identical size (110 kD) from EBV-infected cells; (c) measurement of DNA polymerase activity similar to that of the enzyme induced in EBV-infected cells; and (d) neutralization of the enzymatic activity by the rabbit antiserum and inhibition by phosphonoacetic acid. Our results indicate that the baculovirus expression system provides large quantities of functional polymerase suitable for biochemical and structural analyses, thereby furthering our understanding of the mechanism of viral DNA replication and its inhibition by antiviral drugs.