The colocalization of histamine (HA) and norepinephrine (NE) immunoreactivities was identified within superior cervical ganglia neurons of the guinea pig. HA and NE immunoreactivity levels were significantly attenuated after chemical sympathectomy with 6-hydroxydopamine (6-OHDA). Coexistence of NE and HA was also visualized in the cardiac sympathetic axon and varicosities labeled with anterograde tracer biotinylated dextran amine. Depolarization of cardiac sympathetic nerve endings (synaptosomes) with 50 mM potassium stimulated endogenous HA release, which was significantly attenuated by 6-OHDA or a vesicular monoamine transporter 2 (VMAT2) inhibitor reserpine pretreatments. Compound 48/80, a mast cell releaser, did not affect cardiac synaptosome HA exocytosis. Furthermore, K⁺ evoked HA release was abolished by the N-type Ca²⁺-channel blocker -conotoxin, but was not affected by the L-type Ca²⁺-channel blocker lacidipine. Cardiac synaptosome HA exocytosis was augmented by the enhanced synthesis of HA or the inhibition of HA metabolism. HA H₃-receptor activation by (R)--methylhistamine inhibited high K⁺-evoked histamine release. The HA H₃ receptor antagonist thioperamide enhanced K⁺-evoked HA release and blocked the (R)--methylhistamine effect. The K⁺-evoked endogenous NE release was attenuated by preloading the cardiac synaptosomes with L-histidine or quinacrine. These inhibitory effects were reversed by thioperamide or antagonized by -fluoromethylhistidine. Our findings indicate that high K⁺-evoked co-release of NE and HA may be inhibited by endogenous HA via activation of presynaptic HA H₃-receptors. The H₃-receptor may function as an autoreceptor, rather than a heteroreceptor, in the regulation of sympathetic neurotransmission, and HA may be a novel sympathetic neurotransmitter.