Human intestinal trefoil factor, hITF, a secretory polypeptide found mainly in the human gastrointestinal tract, is a member of the newly characterized trefoil factor or P-domain peptide family representing putative growth factors. Here we describe the identification of this gut peptide in the human brain and pituitary. With reverse transcriptase polymerase chain reaction, we were able to isolate and clone the transcript from human hypothalamus. An antibody generated against a synthetic peptide derived from the carboxyl terminus of hITF was used for immunohistochemical studies of appropriate tissue sections. Neurons expressing hITF were identified in two magnocellular hypothalamic nuclei, the paraventricular and periventricular nuclei. hITF polypeptide was also observed in Herring bodies of the neurohypophysis and in secretory cells of the adenohypophysis. Double immunostaining with antigrowth hormone antibody showed partial coexistence in a selected subpopulation of adenohypophysial cells. Localization of hITF in the hypothalamo-neurohypophysial system may suggest a modulatory action on the classical magnocellular nonapeptides vasopressin and oxytocin, and further indicates an adenohypophysial importance of this peptide. It is likely that hITF represents a novel neuropeptide of yet unknown function.