In patients with Kaposi’s sarcoma (KS), human herpesvirus 8 (HHV-8) can invariably be detected in KS tumor tissue and, at a lower frequency, in prostate tissue and peripheral blood B lymphocytes. Whereas the majority of KS spindle cells are latently infected by HHV-8, linear HHV-8 genomes characteristic for lytic infection are found predominantly in the peripheral blood cells of KS patients. In this study, we show that HHV-8 can stably infect B lymphocytes in vitro in the presence of Epstein-Barr virus (EBV). We were able to generate immortalized HHV-8⁺/EBV⁺ lymphoblastoid cell lines (LCLs) derived from peripheral blood mononuclear cells (PBMC) of EBV⁻ and EBV⁺ donors. In HHV-8⁺/EBV⁺ LCLs, which have the phenotype of activated B lymphocytes (CD19⁺, surface immunoglobulin M, CD23⁺, CD30⁺, CD80⁺), HHV-8 was still present after more than 25 passages (more than 9 months of culture). Latent viral transcripts and proteins were present in nonstimulated HHV-8⁺/EBV⁺ LCLs. After induction by phorbol ester and n-butyrate, HHV-8⁺/EBV⁺ LCLs expressed lytic HHV-8 transcripts and proteins. Moreover, HHV-8 could be serially passaged from HHV-8⁺/EBV⁺ LCLs to fresh PBMC.