The interaction of the potent histamine H3-receptor ligands i.e. (R)alpha-methylhistamine, an agonist, and thioperamide, an antagonist, with the three classes of cerebral histamine receptors was studied in vitro and in vivo. The histamine-induced stimulation of 3',5'-cyclic AMP accumulation in slices of guinea-pig hippocampus was not modified by thioperamide (up to 0.1 mM) and (R)alpha-methylhistamine stimulated cyclic AMP accumulation only at millimolar concentrations. Hence, both (R)alpha-methylhistamine and thioperamide were at least 100,000-fold more potent at H3- than at H1- or H2-receptors in brain. In vivo, the turnover of histamine in rat cerebral cortex, as determined from its depletion elicited by alpha-fluoromethylhistidine in a synaptosomal fraction was not modified by mepyramine and zolantidine but was markedly enhanced by thioperamide at a low dose (ED50 = 2 mg/kg). Thioperamide also elicited a long-lasting decrease in synaptosomal histamine and increase in radioimmunoassayable N tau-methylhistamine. In contrast, (R)alpha-methylhistamine markedly reduced cortical [3H]histamine synthesis (ED50 = 5 mg/kg). This long-lasting action was accompanied by an increase in synaptosomal histamine and a decrease in N tau-methylhistamine levels. These changes were compared with those in plasma drug levels. Hence the two H3-receptor ligands appear to modify the activity of cerebral histamine neurons markedly and in a long-lasting and opposite manner.