Kaposi's sarcoma-associated herpesvirus (KSHV) or human herpesvirus 8 (HHV-8) is a gamma-herpesvirus consistently identified in Kaposi's sarcoma (KS), primary effusion lymphoma, and multicentric Castleman's disease. KSHV infection appears to be necessary, but not be sufficient for development of KS without other co-factors. However, factors that facilitate KSHV to cause KS have not been well defined. Because patients with KS are often immunosuppressed and susceptible to many infectious agents including human herpesvirus 6 (HHV-6), we investigated the potential of HHV-6 to influence the replication of KSHV. By co-culturing HHV-6-infected T cells with KSHV-latent BCBL-1 cell line, infecting BCBL-1 cells with HHV-6 virions, and generating heterokaryons between HHV-6-infected T cells and BCBL-1 cells, we showed that HHV-6 played a critical role in induction of KSHV replication, as determined by production of lytic phase mRNA transcripts and viral proteins. We confirmed and extended the results by using a luciferase reporter assay in which KSHV ORF50 promoter, the first promoter activated during KSHV replication, drove the luciferase expression. Besides HHV-6, we also found that cytokines such as interferon-gamma partially contributed to induction of KSHV replication in the co-culture system. These findings suggest that HHV-6 may participate in KS pathogenesis by promoting KSHV replication and increasing KSHV viral load.