Chronic fatigue syndrome (CFS) has been widely studied by neuroimaging techniques in recent years with conflicting results. In particular, using single-photon emission computed tomography (SPECT) and perfusion tracers, hypoperfusion has been found in several brain regions, although the findings vary across research centers. The objective of this study was to investigate brain metabolism of patients affected by CFS, using [¹⁸F]fluorine-deoxyglucose (¹⁸FDG) positron emission tomography (PET). We performed ¹⁸FDG PET in 18 patients who fulfilled the criteria of the working case definition of CFS. Twelve of the 18 patients were females; the mean age was 34 ± 15 years (range, 15–68) and the median time from CFS diagnosis was 16 months (range, 9–138). Psychiatric diseases and anxiety/neurosis were excluded in all CFS patients. CFS patients were compared with a group of 6 patients affected by depression (according to DSM IV-R) and 6 age-matched healthy controls. The CFS patients were not taking any medication at the time of PET, and depressed patients were drug-free for at least 1 week before the PET examination. The PET images examined 22 cortical and subcortical areas. CFS patients showed a significant hypometabolism in right mediofrontal cortex (P = 0.010) and brainstem (P = 0.013) in comparison with the healthy controls. Moreover, comparing patients affected by CFS and depression, the latter group showed a significant and severe hypometabolism of the medial and upper frontal regions bilaterally (P = 0.037–0.001), whereas the metabolism of brain stem was normal. Brain ¹⁸FDG PET showed specific metabolism abnormalities in patients with CFS in comparison with both healthy controls and depressed patients. The most relevant result of our study is the brain stem hypometabolism which, as reported in a perfusion SPECT study, seems to be a marker for the in vivo diagnosis of CFS.