In contrast to the corresponding mouse and rat orthologs, the human histamine H-4 receptor (hH(4)R) shows extraordinarily high constitutive activity. In the extracellular loop (ECL), replacement of F169 by V as in the mouse H4R significantly reduced constitutive activity. Stabilization of the inactive state was even more pronounced for a double mutant, in which, in addition to F169V, S179 in the ligand binding site was replaced by M. To study the role of the FF motif in ECL2, we generated the hH(4)R-F168A mutant. The receptor was coexpressed in Sf9 insect cells with the G-protein subunits G alpha(12) and G beta(1)gamma(2), and the membranes were studied in [H-3] histamine binding and functional [S-35] GTP gamma S assays. The potency of various ligands at the hH(4)R-F168A mutant decreased compared to the wild-type hH(4)R, for example by 30-and more than 100-fold in case of the H4R agonist UR-PI376 and histamine, respectively. The high constitutive activity of the hH(4)R was completely lost in the hH(4)R-F168A mutant, as reflected by neutral antagonism of thioperamide, a full inverse agonist at the wild-type hH(4)R. By analogy, JNJ7777120 was a partial inverse agonist at the hH(4)R, but a partial agonist at the hH(4)R-F168A mutant, again demonstrating the decrease in constitutive activity due to F168A mutation. Thus, F168 was proven to play a key role not only in ligand binding and potency, but also in the high constitutive activity of the hH(4)R.