Neuromedin U (NmU) is a novel peptide that potently contracts smooth muscle, including rat uterus in vitro. No receptors have so far been demonstrated in any tissue. We have characterized the receptor for NmU in rat uterine membrane preparations. The binding of [125I] rat NmU was saturable, specific, reversible, and time, temperature, and pH dependent. The nonhydrolysable analog of GTP, GTP-gamma-S, reduced binding in a dose-dependent manner, suggesting that the binding site is coupled to a G-protein. Scatchard analysis of saturation binding studies showed a single class of binding site with a maximal binding capacity of 580 +/- 140 fmol/mg membrane protein and a dissociation constant of 0.35 +/- 0.09 nM (n = 3). Rat NmU and NmU-8 (the C-terminal region of the larger molecule) displaced [125I]rat NmU, displaying an IC50 of 1 x 10(-9) M and 6 x 10(-8) M, respectively. Chemical cross-linking studies demonstrated NmU binding sites on the rat uterus to have an M(r) of 48,500. This band was absent in the presence of 0.2 microM NmU. Thus the rat uterus contains specific NmU binding sites through their physiological role awaits identification.