Equilibrium binding studies showed that butyrate-treated HT-29 cells express a high-affinity 125 I-labeled peptide YY ( 125 I-PYY) binding site with a dissociation constant of 0.32 ± 0.12 nM (mean ± SE, n = 4). This site was Y 1 preferring because neuropeptide Y (NPY) and the Y 1 -selective agonist [Leu 31 ,Pro 34 ]NPY were equipotent to PYY at displacing 125 I- PYY; PYY-(13-36) and pancreatic polypeptide were >1,000- and 10,000-fold less potent at displacing the radioligand. PYY and [Leu 31 ,Pro 34 ]NPY inhibited forskolin-stimulated adenosine 3',5'-cyclic monophosphate production 63% and 48%, respectively, with a half-maximal inhibitory concentration between 0.1 and 1.0 nM. PYY and [Leu 31 ,Pro 34 ]NPY had no effect on release of intracellular calcium done or on the increase in intracellular calcium concentration caused by carbachol or neurotensin. Northern blot analysis of poly(A) + RNA from HT-29 cells demonstrated a single transcript of 2.5 kb that hybridized to a human Y 1 -receptor cDNA probe. Sequence analysis of a reverse transcription-polymerase chain reaction product amplified with primers based on human Y 1 -receptor cDNA confirmed that these cells contained mRNA encoding the human Y 1 receptor. These studies show that butyrate- treated HT-29 cells constitutively express the Y 1 -preferring NPY/PYY receptor and Y 1 mRNA and provide a new model for studies of PYY-regulated epithelial cell function and tissue-specific expression of the human Y 1 - receptor gene.