A large proportion (30%) of tissue glutaminase activity was found localized in synaptosome fractions as well as purified mitochondrial fractions, where it is also enriched on a protein basis (2-fold). Sulphate was more effective (2-8-fold) than phosphate or chloride in activating the enzyme at concentrations above 10-12 mM, but equivalent to phosphate at lower concentrations (1-3 mM). At CSF levels (0.5 mM), glutamine is readily used by synaptosomes as a substrate in the presence of 10 mM glucose. It constitutes at least 50% to the carbon of aspartate, glutamate and GABA. Pool sizes of these amino acids are maintained in the presence of glutamine and glucose at CSF levels, but not by glucose alone. Ammonium ion at 1 mM substantially (30%) inhibits glutaminase utilisation by synaptosomes. It is suggested that CSF glutamine is a major substrate for the nerve ending in situ.