Simian virus 40 (SV40) is known to induce primary brain tumors and lymphomas in animal models. Recently, it was also associated with the pathogenesis of human non-Hodgkin's lymphomas. In the present study, we investigated primary central nervous system lymphomas (PCNSL), a defined subgroup of diffuse large B-cell lymphoma confined to the central nervous system, for the presence of SV40 DNA. Frozen tissue samples of 23 PCNSL derived from human immunodeficiency virus-negative patients were analyzed by two different, fully nested polymerase chain reaction protocols. SV40 DNA sequences could not be detected in any of these samples. Thus, SV40 can be added to the list of viruses that have already been excluded as pathogenetically relevant cofactors in PCNSL.