Central or peripheral administration of the alpha 2-adrenoceptor agonist clonidine causes marked hyperglycemia in the rat. It is not clear whether this effect is mediated within the brain at either pre- or postsynaptic alpha 2-adrenoceptors or whether it is due to peripheral alpha 2-agonist actions. We employed computerized mass spectrometry to measure noradrenaline (NA) and its primary neuronal metabolite 3,4-dihydroxyphenylglycol (DHPG) in the medial basal hypothalamus of rats treated acutely with clonidine, the alpha 2-antagonist yohimbine, the postganglionic noradrenergic blocker guanethidine and the neuroglycopenic agent 2-deoxy-D-glucose (2-DG). That clonidine's hyperglycemic effect was due, in part, to an action at central alpha 2-adrenoceptors was indicated by the ability of guanethidine to significantly inhibit the glucose response. Because of clonidine's inhibition of hypothalamic NA release (assessed by the DHPG/NA ratio), presumably by presynaptic agonism, these data indicated that postsynaptic receptor stimulation by clonidine was involved in activating glucose release. Yohimbine markedly increased the hypothalamic DHPG/NA ratio, reflecting presynaptic stimulation of NA release, but at the same time inhibited the hyperglycemic response due to 2-DG administration. This latter effect to block hyperglycemia is consistent with antagonism of postsynaptic alpha 2-adrenoceptors involved in mediating hepatic glucose output. These data indicate a major role for postsynaptic alpha 2-adrenoceptors in glucoregulation.