Epidemiological studies strongly suggest that a newly discovered herpesvirus, Kaposi's sarcoma associated-herpesvirus (KSHV/HHV8), is the likely infectious cause of Kaposi's sarcoma (KS). Identification of this agent suggests the possibility that existing anti-herpesvirus chemotherapeutics have activity against KSHV. Using KSHV/Epstein-Barr virus (EBV)-coinfected cell line BC-1 and KSHV-infected/EBV-negative cell line BC-3, the effect of DNA polymerase inhibitors in the presence of virus-inducing agents was examined. The phorbol ester TPA induced 8.2-fold EBV replication in BC-1 cells with only minimal concurrent KSHV genome replication in BC-1, but induced fourfold KSHV in BC-3 cells. TPA, however, induced transcripts encoded by the lytic cycle major capsid protein gene that were inhibited by both phosphonoacetic acid and phosphonoformic acid either in the KSHV/EBV-infected cell line or in the EBV-negative/KSHV-infected cell line. Transcripts hybridizing to a KSHV-encoded cyclin gene were unaffected by either TPA or DNA polymerase inhibitors in both cell lines. These results show in vitro activity of DNA polymerase inhibitors on KSHV lytic transcript accumulation and may provide a simple assay for evaluating the efficacy of potential anti-KSHV chemotherapeutics.