The properties of DNA polymerases induced by two human herpesviruses, herpes simplex virus type-1 (HSV-1) and Epstein-Barr virus (EBV), have been compared. The HSV-1 and EBV polymerases can be distinguished from one another by differences in the elution profiles in phosphocellulose and single-stranded DNA cellulose columns. Although both enzymes require monovalent cations for optimum activity, the HSV-1 enzyme requires ammonium sulfate whereas the EBV enzyme activity is inhibited by it; on the other hand, the EBV polymerase requires KCl. Other reaction requirements are also different for the two viral enzymes. Thus, when the EBV DNA polymerase was assayed under conditions optimum for the HSV-1 DNA polymerase, only 15% of its activity was expressed. Differences were also noted in sensitivities of the two viral enzymes to the 5'-triphosphates of nucleoside analogs with antiherpesvirus activity such as BVdU, IVdU, ACV, FIAC and IdUrd. The HSV-1 polymerase was more sensitive than the EBV DNA polymerase to inhibition by phosphonoacetate, phosphonoformate, aphidicolin and N-ethylmaleimide. However, the EBV DNA polymerase was more sensitive than HSV-1 DNA polymerase to heat treatment at 42 degrees C. Thus, the marked differences between the two viral enzymes can be useful in identifying enzyme activities in cells producing the virus and also in studying the biochemical mechanism of action of some of the antiviral agents.