The effects of stimulation of the ventromedial hypothalamus (VMH) or lateral hypothalamus (LH) with potassium chloride through a microdialysis probe were studied. The concentrations of ACh and norepinephrine (NE) in the dialysate obtained from the hypothalamic nuclei and plasma glucose concentration were measured. Stimulation of the hypothalamic nuclei, VMH and LH, with potassium increased the plasma glucose level as well as the extracellular concentrations of ACh and choline. Addition of atropine, a muscarinic ACh receptor antagonist, into the potassium solution reduced the increase in the level of plasma glucose. Cholinergic stimulation of these nuclei with neostigmine increased the extracellular concentrations of ACh and plasma glucose. Stimulation of the nuclei with potassium also increased the release of NE. However, stimulation of the VMH or LH with NE and/or pargyline, a monoamine oxidase inhibitor, through the dialysis probe membrane did not significantly increase the plasma glucose concentration. These results suggest that activation of the muscarinic cholinergic or ACh-receptive neurons in the hypothalamic nuclei, VMH and LH, contribute to the elevation of plasma glucose level.