Activation of presynatic histamine H(3) receptors (H(3)R) down-regulates norepinephrine exocytosis from cardiac sympathetic nerve terminals, in both normal and ischemic conditions. Analogous to the effects of alpha(2)-adrenoceptors, which also act prejunctionally to inhibit norepinephrine release, H(3)R-mediated antiexocytotic effects could result from a decreased Ca(2+) influx into nerve endings. We tested this hypothesis in sympathetic nerve terminals isolated from guinea pig heart (cardiac synaptosomes) and in a model human neuronal cell line (SH-SY5Y), which we stably transfected with human H(3)R cDNA (SH-SY5Y-H(3)). We found that reducing Ca(2+) influx in response to membrane depolarization by inhibiting N-type Ca(2+) channels with omega-conotoxin (omega-CTX) greatly attenuated the exocytosis of [(3)H]norepinephrine from both SH-SY5Y and SH-SY5Y-H(3) cells, as well as the exocytosis of endogenous norepinephrine from cardiac synaptosomes. Similar to omega-CTX, activation of H(3)R with the selective H(3)R-agonist imetit also reduced both the rise in intracellular Ca(2+) concentration (Ca(i)) and norepinephrine exocytosis in response to membrane depolarization. The selective H(3)R antagonist thioperamide prevented this effect of imetit. In the parent SH-SY5Y cells lacking H(3)R, imetit affected neither the rise in Ca(i) nor [(3)H]norepinephrine exocytosis, demonstrating that the presence of H(3)R is a prerequisite for a decrease in Ca(i) in response to imetit and that H(3)R activation modulates norepinephrine exocytosis by limiting the magnitude of the increase in Ca(i). Inasmuch as excessive norepinephrine exocytosis is a leading cause of cardiac dysfunction and arrhythmias during acute myocardial ischemia, attenuation of norepinephrine release by H(3)R agonists may offer a novel therapeutic approach to this condition.