Background and Objective Human herpesvirus 8 (HHV-8) has been found in association with Kaposi's sarcomas in human immunodeficiency virus (HIV)-positive and -negative patients, primary effusion lymphomas (PELs), multicentric Castleman's disease, and multiple myeloma. The PELs share several features with acquired immunodeficiency syndrome (AIDS)-associated primary central nervous system lymphomas (1 °CNS-L), including; B-cell phenotype, infection with Epstein-Barr virus, and lack of c-myc gene rearrangements. This prompted us to investigate the role of HHV-8 in 1°CNS-L and other brain lymphomas. Methods To identify HHV-8, we performed nested and single polymerase chain reaction using DNA extracted from autopsy and biopsy brain lymphoma specimens of 36 patients with and without AIDS. Results We detected HHV-8 DNA in 56% of all l°CNS-L, with similar rates for patients with and without AIDS, but in just one of five metastatic B-cell lymphomas. Brain tissues histologically uninvolved by lymphoma were negative for HHV-8 in three patients in whom their 1°CNS-L was HHV-8 positive. In contrast, we easily demonstrated HHV-8 DNA in spleens and a KS lesion of patients with HHV-8—positive 1°CNS-L. All non lymphoma brain controls were negative for HHV-8. Conclusions HHV-8 DNA is found in a significant percentage of 1°CNS-L, including patients with and without AIDS. Identification of HHV-8 in splenic tissue and absence of HHV-8 in control brain tissues suggests the possibility of a peripheral lymphocyte reservoir for HHV-8. Systemically infected lymphocytes may play a direct or indirect role in the pathogenesis of 1°CNS-L.