Radiolabelled glutamine and glucose were infused into lateral ventricles of rats in order to label transmitter amino acid pools in vivo. Brain regions close to the lateral ventricle (hippocampus, corpus striatum, hypothalamus) were labelled more effectively than more distant structures such as cerebral cortex or cerebellum. All regions were labelled to much the same extent over 30-150 min by [U-14C]glucose, [U-14C]glutamine, or [3H]glutamine administered alone or together in double-label experiments when allowance was made for any differences in precursor specific radioactivities. Slices of cerebral cortex or hippocampus from brains labelled in vivo were incubated and stimulated in vitro with veratrine (75 microM); tetrodotoxin (1 microM) was present in the control medium. Single-label experiments showed that [U-14C]glutamine was more effective than [U-14C]glucose for labelling releasable glutamate and GABA. Double-label experiments showed that [3H]glutamine and [U-14C]glucose given together in vivo labelled glutamate and GABA releasable in vitro to a similar extent. Both types of experiment emphasise the large contribution made by glutamine in vivo to pools of transmitter glutamate and GABA.