We conducted this study to determine whether infection with human herpesvirus (HHV) 6A, HHV-6B, or HHV-7 differed between patients with chronic fatigue syndrome and control subjects. We recruited 26 patients and 52 nonfatigued matched control subjects from Atlanta. Serum samples were tested by enzyme immunoassay for seroreactivity to HHV-6, and all were seropositive. Lymphocyte specimens were cocultivated with cord blood lymphocytes and assayed for HHV-6 and HHV-7; neither virus was isolated. Finally, lymphocytes were tested by use of 3 polymerase chain reaction methods for HHV-6A, HHV-6B, and HHV-7 DNA. HHV-6A or HHV-6B DNA was detected in 17 (22.4%) of 76 samples, and there were no significant differences (by matched analyses) between patients (3 [11.5%] of 26) and control subjects (14 [28%] of 50). HHV-7 DNA was detected in 14 subjects, and although control subjects (12 [24%]) were more likely than patients (2 [7.7%]) to be positive, the difference was not statistically significant. We found no evidence that active or latent infection with HHV-6A, HHV-6B, HHV-7, or any combination these 3 HHVs is associated with chronic fatigue syndrome.