Peripheral blood mononuclear cells collected from 13 patients with chronic fatigue syndrome and 13 healthy controls were analyzed for the presence of human herpesvirus 6 (HHV-6) DNA by variant-specific polymerase chain reaction and dot blot hybridization. HHV-6 DNA was detected in 7 of 13 (53%) patients, and of those 7 patients, 4 were positive for HHV-6 variant A DNA and 3 were for variant B. No HHV-6 DNA was detected in the controls. Serum antibody titers to the late antigen and antibody prevalence to the early antigen of HHV-6 were significantly higher in the patient group. These results suggest active replication of HHV-6 in patients with chronic fatigue syndrome.