Although constitutive activity was shown to occur with many recombinant and/or mutated G-protein-coupled receptors, the physiological relevance of the process has remained debated. We have further explored this important issue with the histamine H3 receptor (H3R), a presynaptic receptor regulating histamine neuron activity in the brain. Constitutive activity of the recombinant receptor was studied using [3H]arachidonic acid release, [35S]GTPγ[S] binding and inhibition of cAMP accumulation. Evidence for constructive activity was obtained in these three functional assays with two isoforms of the rat H3 receptor, as well as with the human H3 receptor, expressed at physiological densities. Several standard H3-receptor antagonists, such as thioperamide and ciproxifan, were in fact acting as potent inverse agonists. Proxyfan opposed both agonists and inverse agonists and was therefore identified as a neutral antagonist. Using these drugs, we show high constitutive activity of native receptors. [35S]GTPγ[S] binding demonstrated constitutive activity of H3 receptors expressed at a normal level in mouse or rat brain. Constitutive activity of presynaptic H3 autoreceptors modulates histamine release from cortical synaptosomes in vitro and controls histamine neuron activity in vivo. This implies that inverse agonists rather than neutral antagonists may find therapeutic applications.