12-0-Tetradecanoyl phorbol-13-acetate (TPA) has been widely known as an activator of Epstein-Barr Virus (EBV) replication and is a direct activator of protein kinase C (PKC). These facts suggest that EBV DNA synthesis might at least partly be dependent upon PKC activity. In this report, the effects of two different types of PKC inhibitors on EBV DNA synthesis were investigated by slot blot hybridization using a biotin-labeled probe. Staurosporine and H-7, inhibitors acting on the catalytic domain of PKC, prevented the growth reduction of P3HR-1 cells harboring EBV genomes and the induction of viral DNA synthesis by TPA. Calphostin C and sphingosine, which have been reported to suppress the enzyme activity by acting on the regulatory domain of PKC, did not exert efficiently effects on cellular growth and viral replication at increasing concentrations of TPA. From these results it is suggested that PKC is involved in the control of viral DNA synthesis in P3HR-1 cells and that for the inhibition of virus growth, it is more efffective to suppress the activity of the catalytic domain of this enzyme than acting on the regulatory domain and competing with PKC activators such as TPA for binding.