Primary central nervous system lymphomas (PCNSL) are derived from germinal center B cells. Recent molecular studies indicate that the tumor cells or their precursors have experienced antigenic stimulation. Attractive candidates for such antigens are pathogens with the capacity to reside in the brain. The aim of the present study was to evaluate whether human herpes virus (HHV)-8 is involved in the pathogenesis of PCNSL. A series of 46 PCNSL, 31 from HIV-negative and 15 from HIV-positive patients, were analyzed using various molecular biological and immunological approaches. Nested PCR with two different protocols unequivocally demonstrated that PCNSL from HIV-negative patients did not harbor HHV-8 DNA. Among AIDS-associated PCNSL, HHV-8 DNA was found in only 1 tumor. In situ hybridization studies revealed that the lymphoma cells were HHV-8 negative in all cases. Single small mononuclear cells, most likely corresponding to bystander lymphocytes, were identified as the cellular source of HHV-8 in the HIV-positive patient with an HHV-8 PCR signal. These studies largely rule out a role for HHV-8 in the pathogenesis of PCNSL in both HIV-negative as well as HIV-positive patients.