Synaptic vesicle fractions have been isolated from cerebral cortex, subcortical telencephalon, whole brain and spinal cord by density gradient centrifugation. The Mg2+ ATP-dependent vesicular uptake and the Na(+)-dependent synaptosomal uptake of glycine, GABA and L-glutamate has been compared in the different regions. All these regions contain GABA as inhibitory neurotransmitter, whereas glycine only plays a dominant role as such in the spinal cord. The ratio between GABA and glycine uptake in the different vesicle fractions was similar, and the ratios differed greatly from the ratios in the synaptosomal uptake. In contrast, the ratio between glutamate and GABA uptake in vesicles from different regions differed, and these ratios corresponded to the ratios in the synaptosomal uptake. These results indicate that glycine is taken up into synaptic vesicles from non-glycine terminals, and we suggest that GABA and glycine can be taken up into the same vesicle population.