Human herpesvirus type 8 (HHV-8) has been proposed as a pathogenetic factor for immunosuppression-associated primary central nervous system lymphoma (PCNSL). To verify this hypothesis, HHV-8 infection was investigated in 31 persons with PCNSL (16 AIDS-related, 15 AIDS-unrelated) and in 30 persons with systemic B cell non-Hodgkin's lymphomas (B-NHL; 15 AIDS-related, 15 AIDS-unrelated). All subjects with PCNSL scored negative by single-step polymerase chain reaction (PCR), suggesting a tumor virus load of <100 viral copies/200,000 human haploid genome equivalents (HHGE). By applying Poisson assumptions to nested PCR, 16 of 31 persons with PCNSL were devoid of HHV-8 sequences: 1 subject with AIDS and PCNSL had 1-100 viral copies/200,000 HHGE, and 14 with PCNSL had <1 viral copy/200,000 HHGE. Similarly, 10 of 30 persons with systemic B-NHL were devoid of HHV-8 sequences; 20 had <1 viral copy/200,000 HHGE. The extremely low levels of infection rule out a role of HHV-8 in PCNSL pathogenesis and are consistent with HHV-8 infection of bystander cells contaminating the tumor clone.