In our previous studies, we have identified that histamine (HA) is co-released with noradrenaline (NA) from the sympathetic nerve terminals and acts as a novel sympathetic neurotransmitter. However, the modulation of sympathetic HA release by sympathetic prejunctional receptors is still unknown. Therefore, in this study, we explored the modulation effect of alpha(2) adrenoceptors on sympathetic HA release and the interaction of alpha(2) adrenoceptors with HA H(3) receptors in vas deferens isolated from guinea pig. The selective alpha(2) adrenoceptor agonist brimonidine decreased the HA overflow in a concentration-dependent manner and abolished the contractile responses mediated by sympathetic HA release. Yohimbine competitively blocked the effect of brimonidine on HA release. Similarly, the HA H(3) receptor agonist R-(alpha)-methylhistamine also decreased HA release, and thioperamide blocked the effect of R-(alpha)-methylhistamine. When used singly, both yohimbine and thioperamide facilitated HA release. In addition, the inhibitory effect of brimonidine on HA release was stronger in the presence of thioperamide, while it was reduced when HA H(3) receptors were activated by R-(alpha)-methylhistamine. Our findings indicate for the first time that sympathetic HA release is negatively modulated both by prejunctional alpha(2) receptors and HA H(3) receptors and that these two prejunctional receptors interact with each other in modulating sympathetic HA release.