GT-2016, a non-thiourea-containing imidazole, has been developed as a histamine H3 antagonist. In vitro and in vivo studies in rats were conducted to characterize receptor selectivity and autoreceptor functionality for GT-2016. GT-2016 demonstrated high affinity (43.8 +/- 3.0 nM) and selectivity for the histamine H3 receptor in vitro. In vivo, GT-2016 (3, 10 and 30 mg/kg i.p. and p.o.) was shown to cross the blood-brain barrier and dose-dependently bind to cortical histamine H3 receptors. GT-2016 induced dose-dependent increases in histamine turnover at concentrations that exhibited significant histamine H3 receptor occupancy. Also, in vivo microdialysis experiments were conducted in awake, freely moving rats treated with GT-2016. GT-2016 (10 and 30 mg/kg i.p.) increased histamine release by approximately 75% above baseline within 1 hr, and elevated histamine release was observed for up to 2.5 hr after the higher dose. In contrast, GT-2016 was devoid of activity on histamine methyltransferase in vitro at concentrations up to 3 microM. Taken together, the results show that GT-2016 crosses the blood-brain barrier, binds to H3 receptors and increases the release of histamine in the cerebral cortex, consistent with blockade of presynaptic H3 autoreceptors. In summary, these findings allowed us to identify and characterize the in vitro and in vivo biochemical properties of a novel H3 receptor antagonist, GT-2016.