[35S]GTPγS autoradiography is a novel functional approach to detect G protein activity in brain cryostat sections in response to stimulation of G protein-coupled receptors (GPCRs). One immediate advantage of this approach is that for the first time receptor signalling can be studied in anatomically defined brain structures. A salient feature is the heterogeneously distributed signal in many regions under basal assay conditions. Endogenous adenosine, acting via A1 receptors, is so far the only identified factor contributing to this. Despite elimination of the adenosine signal, several “hot spot” loci (e.g. the hypothalamus) representing high local basal G protein activity persist, awaiting for the identification of the endogenous ligands responsible for this activity. [35S]GTPγS autoradiography predominantly detects signalling of receptors that couple to the Gi class of G proteins, and some 20 distinct receptors belonging to this category have been studied to date under various experimental settings. Although inactivation of Gi, the predominant class of brain G proteins, by N-ethylmaleimide largely eliminates responses to all studied receptors, such treatment does not facilitate detection of responses to Gs- or Gq-coupled receptors. [35S]GTPγS autoradiography is particularly well-suited for functional studies on brain lysophospholipid receptors, for which conventional radioligand binding assays are not available. [35S]GTPgS autoradiography has facilitated the discovery of novel GPCRs, such as the P2Y12 receptor, in the nervous system. Additional applications are also emerging. It is evident that the integrity of the basic signalling unit, which for an increasing number of GPCRs resides in specialized membrane microdomains, is better preserved in cryostat sections than in bulk membrane preparations, suggesting that controversial issues, such as constitutive receptor activity in native brain tissue, can be rigorously addressed using [35S]GTPγS autoradiography. Perhaps we are only beginning to appreciate the true potential of this approach for the neuropharmacological studies on the divergent family of GPCRs.