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['251]Monoiodinated vasoactive intestinal peptide (1251-VIP) was cross-linked with human colonic adenocarcinoma cells (HT 29 cells) grown as a monolayer using dithiobis(succinimidy1propionate) as cross-linking reagent. The cross-linked polypeptides were separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate.A major polypeptide of M , = 67000 was characterized and it behaved like a high-affinity binding site for VIP according to the following data.1. The concentration of native VIP (0.5 nM) giving half-maximum inhibition of '251-VIP covalent crosslinking with this polypeptide was very similar to that giving half-maximum displacement of '*'I-VIP on HT 29 cells (0.6 nM).2. Glucagon or insulin was unable to inhibit the labelling of the M,-67000 component.3. In our experimental conditions neither specific '2'I-V1P binding nor covalent labelling was observed with monolayers of Madin Darby canine kidney epithelial cells (MDCK cells) or African green monkey kidney fibroblasts (Vero cells) while the M,-67 000 polypeptide was also characterized with human rectal adenocarcinoma cells (HRT 18 cells), known to possess the VIP receptor.4. Preincubation of HT 29 cells with native VIP at 37"C, before 12'I-VIP binding and subsequent crosslinking reaction, decreased the labelling of the M,-67000 polypeptide up to 80%.Assuming one molecule of 1251-VIP cross-linked per polypeptide, we have characterized, for the first time, a major polypeptide of M , = 64000, which belongs to the high-affinity VIP binding site of an intestinal human cell line.A vasoactive substance, already discovered in lung extract, has been isolated from porcine intestine [l] and named vasoactive intestinal peptide (VIP). VIP is a single-chain polypeptide of' 28 residues ( M , = 3326) and belongs to the so-called secretin family, which includes glucagon, gastric inhibitory polypeptide, growth-hormone-releasing factor and PHI/ PHM-27 [2,3]. (PHI-27 and PHM-27 are peptides of 27 amino acids, both with N-terminal His, having C-terminal isoleucine amide and methionine amide respectively.) VIP exhibits the general properties of a neurotransmitter with multiple functions [4, 51. The initial event of the action of VIP is its interaction with a specific membrane receptor at the surface of a target cell. An initial description of VIP receptors has been obtained using rat fat cell plasma membranes [6] intestinal epithelium but not in liver cells where VIP elicit a very low response in term of cAMP production [9].The characterization of the VIP receptor and its purification is a prerequisite to understand further the mechanism of VIP action. Cross-linking reagents have proved useful tools to study the quaternary structure of membrane proteins [lo] and to identify cell surface receptors [ll]. The molecular identification of the VIP receptor in rat intestinal epithelial cell membranes [12] and in rat liver cell membranes [13] has been reported after experiments using such bifunctional reagents. Different polypeptides have been cross-lin...