1 Various histamine derivatives were investigated at the human H-3 receptor (H3R) and H-4 receptor (H4R) stably expressed in human embryonic kidney (HEK)-293 cells using [I-125] iodoproxyfan and [H-3] histamine binding, respectively. 2 In Tris buffer, [H-3] histamine binding to membranes of HEK(hH(4)R) cells was monophasic (K-D of 3.8 +/- 0.8 nM). In phosphate buffer, the Hill coefficient was decreased (n(H) = 0.5 +/- 0.1) and a large fraction of the binding was converted into a low-affinity component (K-D = 67 +/- 27 nM). 3 The inhibition of [H-3] histamine binding by two agonists, a protean agonist and five antagonists/ inverse agonists confirms that the potency of many H3R ligands is retained or only slightly reduced at the H4R. 4 Histamine derivatives substituted with methyl groups in alpha, beta or N-alpha position of the side chain retained a nanomolar potency at the H3R, but their affinity was dramatically decreased at the H4R. With relative potencies to histamine of 282 and 0.13% at the H3R and H4R, respectively, (+/-)- alpha,beta-dimethylhistamine is a potent and selective H3R agonist. 5 Chiral alpha-branched analogues exhibited a marked stereoselectivity at the H3R and H4R, the enantiomers with a configuration equivalent to L-histidine being preferred at both receptors. 6 The methylsubstitution of the imidazole ring was also studied. The relative potency to histamine of 4-methylhistamine (4-MeHA) at the H4R (67%) was similar to that reported at H-2 receptors but, owing to its high affinity at the H4R (K-i = 7.0 +/- 1.2 nM) and very low potency at H-1- and H-3-receptors, it can be considered as a potent and selective H4R agonist. 7 On inhibition of forskolin-induced cAMP formation, all the compounds tested, including 4-MeHA, behaved as full agonists at both receptors. However, the maximal inhibition achieved at the H4R (similar to - 30%) was much lower than at the H3R ( similar to - 80%). Thioperamide behaved as an inverse agonist at both receptors and increased cAMP formation with the same maximal effect (similar to +25%). 8 In conclusion, although the pharmacological profiles of the human H3R and H4R overlap, the structure - activity relationships of histamine derivatives at both receptors strongly differ and lead to the identification of selective compounds.