The binding of [3H](R)alpha-methylhistamine, a potent and specific agonist at histamine H3 receptors, was investigated with membranes of rat cerebral cortex. In phosphate buffer the specific binding defined with thioperamide, an H3 receptor antagonist, displayed characters of reversibility and saturability with a Bmax of approximately 30 fmol/mg protein. The KD, derived from either dissociation/association rates or saturation kinetics at equilibrium, was approximately 0.5 nM at 25 degrees C. Competition studies indicated that the binding occurred with a stereoselectivity and pharmacological specificity similar to that of functional H3 autoreceptors regulating histamine release in brain slices. However, whereas the potency of antagonists was closely similar in the two assay systems, that of agonists was approximately 10-fold higher in the binding assay. Among antagonists burimamide was the only one to compete with a pseudo-Hill coefficient significantly different from unity (nH = 0.48 +/- 0.03), indicating a possible heterogeneity among binding sites. The presence of 2.6 mM Ca2+, in a modified Krebs-Ringer medium, promoted the conversion of a larger fraction of sites into a low-affinity component with a KD of 16 nM. The presence of guanylnucleotides in the Krebs-Ringer medium with Ca2+ abolished the binding to this low-affinity component whereas in a phosphate buffer only the KD was slightly increased. It is concluded that the H3 receptor, like many other amine receptors, is coupled to its still unidentified effector system via a G-protein and regulated by Ca2+. However, unlike the latter, the H3 receptor is down-regulated by the divalent cation.