Derived from the same prohormone, obestatin has been reported to exert effects on food intake that oppose those of ghrelin. The obestatin receptor, GPR39, is present in brain and pituitary gland. Since the gene encoding those two peptides is expressed also in those tissues, we examined further the possible actions of obestatin in vivo and in vitro. Intracerebroventricular administration of obestatin inhibited water drinking in ad libitum fed and watered rats, and in food and water deprived animals. The effects on water drinking preceded and were more pronounced than any effect on food intake, and did not appear to be the result of altered locomotor/behavioral activity. In addition, obestatin inhibited angiotensin II-induced water drinking in animals provided free access to water and food. Current clamp recordings from cultured, subfornical organ neurons revealed significant effects of the peptide on membrane potential suggesting this as a potential site of action. In pituitary cell cultures, log molar concentrations of obestatin ranging from 1.0 pM to100 nM failed to alter basal growth hormone (GH) secretion. In addition, 100 nM obestatin failed to interfere with the stimulation of GH secretion by GH-releasing hormone or ghrelin, and did not alter the inhibition by somatostatin in vitro. We conclude that obestatin does not act in pituitary gland to regulate GH secretion, but may act in brain to alter thirst mechanisms. Importantly, in rats the effects of obestatin on food intake may be secondary to an action of the peptide to inhibit water drinking.