High affinity binding sites for peptide YY (PYY) have been identified and characterized in plasma membranes prepared from rat jejunal epithelium by studying the kinetics, stoichiometry, and chemical specificity of the interaction of ¹²⁵I-labeled PYY with membranes. Binding of [¹²⁵I]PYY was rapid, saturable, reversible, specific, and depended on temperature, pH, and ionic strength. In optimized steady state conditions of binding (2 h of incubation at 15 C), the degradation of both [¹²⁵I] PYY and binding sites did not exceed 20%. The concentration dependence of PYY binding, determined by adding increasing concentrations of [¹²⁵I]PYY, indicated that specific binding saturated at 2–3 nm peptide. Scatchard analysis revealed a single class of binding sites with a dissociation constant (K_d) of 434 ± (se) 56 pm and a binding capacity of 336 ± 41 fmol/mg protein (n + 11). Identical results were obtained when increasing concentrations of unlabeled PYY were added to a fixed concentration of [¹²⁵I]PYY, indicating that the radioiodinated peptide has the same apparent affinity as native PYY. Peptides structurally unrelated to PYY, such as members of the vasoactive intestinal peptide family, insulin, or cholecystokinin octapeptide, were unable to compete with [¹²⁵I]PYY for binding to membranes. Rat, human, and avian pancreatic polypeptides, which display, respectively, 42%, 47%, and 53% homology with PYY, did inhibit [¹²⁵I]PYY binding but with an approximate or equal to 100,000-fold lower potency than PYY, indicating the strict structural requirement for recognition by PYY binding sites. In contrast, natural or synthetic neuropeptide Y, which has 25 out of 36 amino acids in common with PYY, retained a high affinity for PYY binding sites [only 4.7 ± 1.2 (n + 5) times lower than that of PYY]. Specific [¹²⁵I]PYY binding was particularly high in the upper small intestine and could not be detected in stomach, large intestine, or liver. These findings indicate that rat small intestinal epithelium expresses specific binding sites for the candidate gut hormone PYY that also binds the neuropeptide Y with high affinity, suggesting that the two peptides may regulate the function of small intestinal epithelium, through interaction with a common receptor site. (Endocrinology118: 1910–1917, 1986)