The aim of this study was to determine whether forebrain neurons projecting to the mediodorsal nucleus of the thalamus in rats express glutamate decarboxylase messenger RNA as a marker for GABAergic neurons. Forebrain glutamate decarboxylase messenger RNA-containing neurons that project to the mediodorsal nucleus were identified using a combination of retrograde tracing with Fluoro-Gold and in situ hybridization for the messenger RNA encoding the 67,000 molecular weight synthetic enzyme for GABA. Glutamate decarboxylase messenger RNA-containing afferents to the mediodorsal nucleus were observed in the olfactory tubercle, vertical limb of the diagonal band of Broca, ventral pallidum, sublenticular substantia innominata, globus pallidus, lateral preoptic area, bed nucleus of the stria terminalis and reticular nucleus of the thalamus. The largest proportions of glutamate decarboxylase messenger RNA-containing afferents to the mediodorsal nucleus were observed in the vertical limb of the diagonal band, ventral pallidal parts of the olfactory tubercle and the reticular nucleus of the thalamus. Somewhat fewer glutamate decarboxylase messenger RNA-containing, retrogradely labeled neurons were observed in the subcommissural ventral pallidum and sublenticular substantia innominata. These data suggest that a GABAergic projection from the basal forebrain to the mediodorsal nucleus of the thalamus can influence the function of this nucleus.