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Laboratory rodents made hyperammonemic by infusing ammonia into the blood show symptoms of brain cell swelling and increased intracranial pressure. These symptoms could be caused in part by an increase in brain glutamine formed when brain glutamine synthetase (GS) naturally detoxifies ammonia to glutamine. Previous studies on the Gulf toadfish (Opsanus beta) demonstrated that it is resistant to high ammonia exposure (HAE) (96 h LC(50)=10mM) despite an increase in brain glutamine. This study attempts to resolve whether the resistance of O. beta is mediated by special handling of brain water in the face of changing glutamine concentrations. Methionine sulfoximine (MSO), an inhibitor of GS, was used to pharmacologically manipulate glutamine concentrations, and magnetic resonance imaging (MRI) was used to assess the status of brain water. Ammonia or MSO treatment did not substantially affect blood acid-base parameters. Exposure to 3.5mM ammonium chloride in seawater for 16 or 40 h resulted in a parallel increase in brain ammonia (3-fold) and glutamine (2-fold) and a decrease in brain glutamate (1.3-fold). Pre-treatment with MSO prevented ammonia-induced changes in glutamine and glutamate concentrations. HAE also induced an increase in plasma osmolality (by 7%) which was probably due to a disturbance of osmoregulatory processes but which did not result in broader whole body dehydration as indicated by muscle water analysis. The increase in brain glutamine was not associated with any changes in brain water in toadfish exposed to 3.5 mM ammonia for up to 40 h or even at 10, 20 and 30 mM ammonia consecutively and for one hour in each concentration. The lack of brain water accumulation implies that ammonia toxicity in toadfish appears to be via pathways other than cerebral swelling. Furthermore, toadfish pre-treated with MSO did not survive a normally sub-lethal exposure to 3.5 mM ammonia for 40 h. The enhancement of ammonia toxicity by MSO suggests that GS function is critical to ammonia tolerance in this species.