[(35)S]GTPgammaS autoradiography represents a powerful functional approach to detect receptor-dependent G(i/o) protein activity in anatomically defined brain structures. Inherent to this technique, however, is the notable basal signal evident in several brain regions in the absence of receptor stimulation by exogenously added agonist. In the rat brain, much of this basal labelling derives from tonic activation of adenosine A(1) and lysophosphatidic acid LPA(1) receptors in the gray and white matter regions, respectively. Despite the elimination of the two receptor activities, prominent basal [(35)S]GTPgammaS labelling is still evident in discrete brain structures, possibly reflecting regional enrichment of G(i/o) and/or constitutive receptor activity or the presence of still unknown endogenous ligands activating their orphan receptors. Here, the anatomical distribution of the enhanced basal signal was systematically mapped in brain sections of 4-week-old male Wistar rats. Regions with prominent basal [(35)S]GTPgammaS labelling represented neuroanatomically distinct structures, in particular various thalamic and hypothalamic nuclei. For instance, the paraventricular thalamic nucleus, the bed nucleus of the stria terminalis and the subfornical organ were highly labelled, as were the periaqueductal gray and the nucleus of the solitary tract. Pre-treatment with N-ethylmaleimide (NEM), an alkylating agent preventing all known receptor-driven G protein activity in cryostat sections markedly decreased the basal binding in all examined regions. In preliminary screening, selective antagonists for various brain-enriched G(i/o)-coupled receptors failed to suppress the basal signal in any of the studied regions.