Glutamine synthesis, the major pathway of ammonia detoxification, and the intracellular concentration of organic osmolytes in primary astrocytes and F98 glioma cells were investigated with multinuclear magnetic resonance spectroscopy. Acute exposure to ammonia (3 h incubation with NH4Cl) raised the concentration of glutamine and other amino acids, such as glutamate and aspartate, and decreased myo-inositol, hypotaurine, and taurine concentrations. The loss of these osmolytes was partially reversed by co-treatment with the glutamine synthetase inhibitor, methionine sulphoximine. Glutamate, the precursor of glutamine, is provided by stimulated anaplerotic flux via pyruvate carboxylase and glutamate dehydrogenase activity. Thus, the glutamine increase and myo-inositol decrease observed by in vivo magnetic resonance spectroscopy on patients with hepatic encephalopathy may be due to the disturbed osmoregulation in astrocytes caused by accumulation of glutamine and the subsequent loss of organic osmolytes.