GABA plasma membrane transporters mediate GABA uptake into presynaptic terminals and surrounding glial processes and thus play a key role in shaping the time course and spatial extent of GABA's action. In the present study we have investigated the cellular and subcellular localization of two GABA transporters (1 and 3) in the rat thalamus using affinity-purified polyclonal antibodies. GABA transporter-1 and -3 immunoreactivity, detected with immunoperoxidase and immunofluorescence methods, is present throughout the thalamus in small punctate structures scattered in the neuropil among unlabelled neuronal perikarya. Labelling for GABA transporter-3 is always more intense than that for GABA transporter-1. Astrocytic processes, identified by their immunoreactivity for glial fibrillary acidic protein, express both GABA transporters. Ultrastructural investigations confirm that GABA transporter-1 and -3 labelling is restricted to astrocytes. Labelled astrocytes are adjacent to terminals making either symmetric or asymmetric synaptic contacts, and are close to neuronal profiles that do not form synaptic contacts in the plane of the section. In double-labelled thin sections some GABA transporter-1- or -3-positive astrocytic processes, detected with immunoperoxidase labelling, surround GABA-positive terminals, detected with antibodies to GABA and immunogold labelling. These findings demonstrate that in rat thalamus the GABA uptake system mediated by GABA transporter-1 and -3 is localized exclusively to astrocytes near the synapses and in the neuropil, and absent from GABAergic terminals. Astrocytes play therefore an important role in mediating GABA transmission in the thalamus, compared to cortical regions.