It has been suggested that while the sialogogue pilocarpine elicits salivary secretion by acting directly on acinar cells of the salivary glands, it induces drinking behavior by acting on muscarinic receptors in the central nervous system. To study which brain regions are affected by the peripherally injected pilocarpine, we investigated changes in the numbers of c-Fos immunoreactive cells. The injections increased the numbers of c-Fos immunoreactive cells in the subfornical organ, median nucleus of preoptic area, organum vasculosum of lamina terminalis, paraventricular nucleus and supraoptic nucleus. Intracerebroventricular injection of pilocarpine produced similar changes in the expression of c-Fos immunoreactivity. The increases in immunoreactive expression induced by both the intraperitoneally and intracerebroventricularly injected pilocarpine were suppressed by previous intracerebroventricular injection of the muscarinic receptor antagonist atropine. Electrophysiological experiments using slice preparations and whole cell recordings showed that pilocarpine depolarized the membrane of neurons in the subfornical organ and suppressed the inhibitory GABAergic synaptic currents by a presynaptic action. The results suggest that peripherally applied pilocarpine does not act only on the salivary glands as a sialogogue, but also evokes thirst sensation by acting on the center controlling body fluid balance in the central nervous system.