Neuromedin U is a bioactive peptide first isolated from porcine spinal cord. In this paper, we demonstrate that neuromedin U is the cognate ligand for the orphan G protein-coupled receptor, FM-3, isolated originally as a homologue of neurotensin and growth hormone secretogogue receptors. Neuromedin U induced specific and evident elevation of extracellular acidification rates, arachidonic acid metabolite release, and intracellular Ca(2+) mobilization in Chinese hamster ovary cells expressing human FM-3. In addition, radiolabeled neuromedin U specifically bound to membrane fractions prepared from these cells with high affinity. We subsequently analyzed the tissue distribution of neuromedin U and FM-3 mRNAs in rats using quantitative reverse transcription-polymerase chain reaction. Neuromedin U mRNA was highly expressed in the gastrointestinal tract, and the highest expression was detected in the pituitary gland. On the other hand, FM-3 mRNA was highly expressed in the small intestine and lung, suggesting that neuromedin U plays important roles in these tissues. The identification of a specific and functional receptor for neuromedin U will facilitate studies on their physiological roles and the search for receptor agonists and antagonists.