Glutamate is the most abundant excitatory neurotransmitter in the vertebrate central nervous system. It is widely assumed that neurons using this transmitter derive it from several sources: (i) synthesizing it themselves from alpha-ketoglutarate or aspartate, (ii) synthesize it from glial-derived glutamine, or (iii) take up glutamate from the extracellular space. By use of immunocytochemistry we show that glutamate is abundant in the retinal ganglion and bipolar cells of the rabbit, but that immunoreactivity for glutamate in these neurons is reduced below immunocytochemical detection limits after the specific inhibition of glutamine synthesis in glial cells by D,L-methionine D,L-sulphoximine. GABA immunoreactivity in retinal amacrine cells was also reduced after inhibition of glutamine synthetase but the patterns and densities of immunoreactivity for taurine and glycine were unaffected. Therefore, this experimental paradigm does not induce generalized metabolic changes in neurons or glia. This study demonstrates that some glutamatergic neurons are dependent on the synthetic processes in glia for their neurotransmitter content.