Hyperammonemia increases brain glutamine levels, causes astrocytic swelling, and depresses cerebral blood flow (CBF) responsivity to CO2. Methionine sulfoximine (MSO) inhibition of glutamine synthetase activity, known to be enriched in astrocytes, prevents ammonia-induced increases in brain glutamine and water content. We tested the hypothesis that inhibition of glutamine accumulation restores CBF responsivity to CO2during acute hyperammonemia. Pentobarbital-anesthetized rats treated with either vehicle or MSO (150 mg/kg i.p.) received a 6-hour intravenous infusion of either sodium or ammonium acetate. With subsequent induction of hypercapnia, CBF increased from 113±14 (mean±SEM) to 194±9 ml/min per 100 g in control rats but was unchanged from 107±13 to 79±10 ml/min per 100 g in hyperammonemic rats. Treatment with MSO in hyperammonemic rats restored the CBF response to hypercapnia (from 73±8 to 141±14 ml/min per 100 g). With induction of hypocapnia, CBF decreased from 114+11 to 88±11 ml/min per 100 g in control rats but increased from 112±13 to 142±19 ml/min per 100 g in hyperammonemic rats. Treatment with MSO in hyperammonemic rats did not fully restore the response to hypocapnia but prevented the paradoxical increase in CBF (from 80±8 to 80±8 ml/min per 100 g). In control rats, MSO did not affect CO2responsivity. Treatment with MSO prevented ammonia-induced increases in intracranial pressure. Hyposmotic-induced increases in brain water content and intracranial pressure attenuated the CBF response to hypercapnia but, unlike hyperammonemia, did not attenuate the response to hypocapnia. In contrast to hypercapnia, vasodilation in response to arterial hypotension was intact in hyperammonemic rats. We conclude that the grossly abnormal CBF responsivity to CO2alterations during hyperammonemia is linked to glutamine accumulation rather than ammonia per se. Cerebral edema secondary to glutamine accumulation may contribute in part to abnormal CBF responses, although other aspects of astrocyte dysfunction are likely to be important.