Working conditions such as shift work constitute a well-known risk factor for insomnia and excessive daytime sleepiness complaints. We compared brain gamma-aminobutyric acid (GABA), glutamic acid (Glu), glutamine (Gln), and Glx (Glu+Gln) levels in day-shift versus alternate-shift workers with proton magnetic resonance spectroscopy (1H-MRS) at 3T. The study population consisted of 32 healthy adult volunteers (16day-shift and 16 alternate-shift workers). Each subject underwent MRS conducted using a MEGA-PRESS sequence in the early morning and early evening on the same day. Spectroscopy voxels (3.0cm×3.0cm×3.0cm) were placed in the frontal lobe and parieto-occipital lobe. The GABA/Cr ratio in the frontal lobe was significantly lower for the alternate-shift group than for the day-shift group in the early evening (1.885 vs. 0.875). For the other metabolite ratios (Gln/Cr and Glx/Cr), there were no significant differences between the two groups regardless of morning or evening schedule. Our preliminary finding represents a possible alteration of GABA content in the brain related to an irregular work schedule. © 2010 Elsevier Ireland Ltd.