The organization of key molecules at glutamatergic synapses in the rat cerebellar cortex as analyzed by high resolution immunocytochemical techniques using gold particles as markers. The distinct compartmentation of glutamate and glutamine was consistent with biochemical data indicating an active role of glia in the removal of released glutamate and in the supply of glutamine for de novo synthesis of transmitter glutamate. The presence in glial cells of two different glutamate transporters, GLT1 and GLAST, provided further support of this concept. Both transporters were selectively expressed in glial membranes and occurred at higher densities in glial processes surrounding parallel fiber synapses with spines than in glial processes associated with parallel fiber synapses with dendritic shafts. At the former type of synapse, gold particles signalling GLT1 and GLAST could be found within a few nanometers of the postsynaptic density. The rat cerebellum also contains a homologue (rEAAC1) of the glutamate transporter EAAC1, originally cloned from rabbit, mRNA encoding this transporter was restricted to neurons. The exact localization of the rEAAC1 transporter molecules at cerebellar synapses remains to be determined but immunocytochemical and physiological data from other laboratories suggest that they may be preferentially expressed in postsynaptic membranes. Gold particles representing immunoreactivity for the AMPA receptor subunits GluR2/3 were found along the entire mediolateral extent of the postsynaptic specialization of parallel fiber synapses and were rarely encountered at non-synaptic membranes. The present data show that molecules engaged in signalling at cerebellar glutamatergic synapses are precisely organized, consistent with the requirements for rapid signal transmission and efficient removal and recycling of transmitter.