Chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME) is a neuro-immune disease of uncertain pathogenesis. Human parvovirus B19 infection has been shown to occur just prior to development of the onset of CFS/ME in several cases, although B19 seroprevalence studies do not show any significant differences between CFS/ME and controls. In this study, we analysed parvovirus B19 markers in CFS/ME patients (n=200), diagnosed according to Fukuda CDC criteria, and normal blood donors (n=200). Serum from each subject was tested for anti-B19 VP2 IgM and IgG (by Biotrin ELISA), anti-B19 NS1 IgM and IgG (by immunofluorescence), and B19 DNA (by real-time PCR). CFS/ME patients and normal blood donors had a similar B19 seroprevalence (75 % versus 78 %, respectively). Eighty-three CFS patients (41.5 %) as compared with fourteen (7 %) normal blood donors tested positive for anti-B19 NS1 IgG (chi(2)=64.8; P<0.0001; odds ratio=9.42, CI 5.11-17.38). Of these 83 patients, 61 complained of chronic joint pain, while 22 did not. Parvovirus B19 DNA was detected in serum of 11 CFS patients and none of the controls by Taqman real-time PCR (chi(2)=9.35, P<0.002). Positivity for anti-B19 NS1 IgG was associated with higher expression levels of the human CFS-associated genes NHLH1 and GABPA. As NS1 antibodies are thought to indicate chronic or severe courses of B19 infection, these findings suggest that although the seroprevalence of B19 in CFS patients is similar to controls, the immune control of the virus in these patients may not be efficient.