Expression of the human cytomegalovirus (HCMV) (AD169) DNA polymerase gene under the control of the polyhedrin promoter of Autographa californica nuclear polyhedrosis virus in Spodoptera frugiperda (Sf9) cells has provided a source of highly active CMV DNA polymerase. In extracts from CMV-infected cells, the CMV DNA polymerase is found strongly associated with an additional polypeptide, ICP36. This protein has been identified as the CMV homolog of the herpes simplex virus type 1 UL42 gene product and may have a similar function. We have expressed HCMV DNA polymerase and ICP36 in the same system and demonstrated that they interact to form a stable complex. Moreover, ICP36 functions to stimulate the DNA polymerase activity in a template-dependent manner. We have compared the activity of the recombinant DNA polymerase in the presence and absence of ICP36 on a number of DNA templates and measured the effect of the polymerase inhibitors phosphonoformic acid and acyclovir triphosphate.