Abstract: The effects of an acute intravenous infusion of ammonium acetate on rat cerebral glutamate and glutamine concentrations, energy metabolism, and intracellular pH were measured in vivo with 1H and 31P nuclear magnetic resonance (NMR). The level of blood ammonia maintained by the infusion protocol used in this study (∼ 500 μM, arterial blood) did not cause significant changes in arterial Pco2, Po2, or pH. Cerebral glutamate levels fell to at least 80% of the preinfusion value, whereas glutamine concentrations increased 170% relative to the preinfusion controls. The fall in brain glutamate concentrations followed a time course similar to that of the rise of brain glutamine. There were no detectable changes in the content of phosphocreatine (PCr) or nucleoside triphosphates (NTP), within the brain regions contributing to the sensitive volume of the surface coil, during the ammonia infusion. Intracellular pH, estimated from the chemical shift of the inorganic phosphate resonance relative to the resonance of PCr in the 31P spectrum, was also unchanged during the period of hyperammonemia. 1H spectra, specifically edited to allow quantitation of the brain lactate content, indicated that lactate rose steadily during the ammonia infusion. Detectable increases in brain lactate levels were observed ∼ 10 min after the start of the ammonia infusion and by 50 min of infusion had more than doubled. Spectra acquired from rats that received a control infusion of sodium acetate were not different from the spectra acquired prior to the infusion of either ammonium or sodium acetate. The results reported here support earlier findings that an increased blood ammonia concentration has a pronounced effect on the brain concentrations of two important amino acids, glutamate and glutamine. They also provide in vivo evidence for the absence of a sustained alteration in either brain intracellular pH or in the concentration of high-energy phosphate compounds during a period of acute hyperammonemia. The technique of in vivo NMR spectroscopy permits multiple, simultaneous measurements of important intermediary and energy metabolites in a single animal, in real time, prior to and during the systemic perturbation.