The rate of glutamine synthesis and utilization at steady state was measured in vivo in the brains of hyperammonaemic rats by 15N n.m.r. in combination with biochemical techniques. Rats were given an intravenous 15NH4+ infusion at the rate of 4.8 +/- 0.3 mmol/h per kg body wt. for 3.5 +/- 0.2 h, followed by 14NH4+ infusion at the same rate for an additional 5.1 h (chase period). During the chase period, blood ammonia (0.61 +/- 0.015 mumol/g), brain ammonia (2.9 +/- 0.3 mumol/g), glutamate (9.4 +/- 0.8 mumol/g) and glutamine (15N + 14N; 14.4 +/- 1.3 mumol/g) were at steady state. The rate of change in the cerebral [5-15N]glutamine concentration was measured in vivo by 15N n.m.r. at 20.27 MHz. To estimate 15N enrichment of precursor ammonia for glutamine synthetase (GS) in astrocytes which are interposed between cerebral capillaries and neurons, 15N enrichments of blood and brain ammonia were measured by gas chromatography-mass spectrometry. The in vivo rate of glutamine synthesis, which is equal to the rate of glutamine utilization at steady state, was estimated, from the observed rate of change in [5-15N]glutamine concentration and 15N enrichment of brain glutamine, to be 4.8 +/- 1.1 mumol/h per g of brain if 15N enrichment of ammonia at the site of GS in astrocytes is equal to that of blood-borne ammonia, and 13.0 +/- 3.9 mumol/h per g if it is equal to that measured for the whole brain. The observed GS activity in vivo in the brain of the hyperammonaemic rat is 2-5% of the reported optimum activity in vitro measured at enzyme-saturating concentrations of all substrates. The result suggests that substrates and/or cofactors other than ammonia kinetically limit GS activity in vivo. The g.c. chromatogram and mass spectrum of ammonia-derived N-trifluoroacetyl-dibutylglutamate (TAB-glutamate) are shown in Supplementary Publication SUP 50170 (4 pages), which has been deposited at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire, U.K., from whom copies can be obtained on the terms indicated in Biochem. J.