Chronic Fatigue Syndrome (CFS) is characterized by debilitating fatigue, somatic symptoms and cognitive impairment. An infectious basis has been proposed; candidate agents include enteroviruses, herpesviruses, retroviruses and Borna disease virus (BDV), a novel neurotropic virus associated with neuropsychiatric disorders. Sera and peripheral blood mononuclear cells (PBMC) from Swedish CFS patients were assayed for evidence of infection using ELISA and Western immunoblot for detection of antibodies to BDV proteins N, P and gp18; and using nested reverse transcriptase polymerase chain reaction (RT-PCR) for detection of BDV N- and P-gene transcripts. No specific immunoreactivity to BDV proteins was found in sera from 169 patients or 62 controls. No BDV N- or P-gene transcripts were found through RT-PCR analysis of PBMC from 18 patients with severe CFS. These results do not support a role for BDV in pathogenesis of CFS.