As a test of the hypothesis that elevated titers of viral antibodies in patients with chronic fatigue syndrome (CFS) are due to a nonspecific polyclonal immune response, antibodies to Epstein-Barr virus (EBV), human herpesvirus 6 (HHV-6), and 14 enteroviruses in 20 patients with CFS and 20 age- and gender-matched controls were simultaneously measured. Similarly, titers of IgG to herpes simplex virus (HSV) types 1 and 2 were measured in 18 of these cases and in the respective controls. IgG to EBV viral capsid antigen (VCA) was present at titers > or = 1:320 in 55% of cases vs. 15% of controls (P = .02). The geometric mean titers of early antigen antibody to EBV, HHV-6 IgG, and HSV-1 and HSV-2 IgG were not significantly different among cases and controls. Of the 14 enteroviral antibodies tested for, only those to coxsackieviruses B1 and B4 were present at significant titers (> or = 1:8) in cases vs. controls (P = .02 and P = .001, respectively). Of the cases, 19 (95%) had either an EBV VCA IgG titer > or = 1:320 or a coxsackievirus B1 or B4 antibody titer > or = 1:8, a percentage significantly higher than that of controls (40%; P = .0004). Titers of EBV VCA IgG and coxsackievirus B1 and B4 antibodies were simultaneously elevated in only 20% of cases. There was no correlation between elevated titers of EBV VCA IgG and IgG to HHV-6, HSV-1, and HSV-2 or antibody to coxsackieviruses B1 and B4 in the cases. The prevalence of reported allergies to medications or other substances was identical in both groups (60%). These findings suggest that in the majority of cases of CFS, elevation of viral antibody titers is not due to a nonspecific polyclonal immune response.