BACKGROUND Vasoactive intestinal polypeptide (VIP) may be a nonadrenergic, noncholinergic inhibitory transmitter in the lower esophageal sphincter (LES). There is no biochemical evidence of VIP receptors in the LES. METHODS Using membranes from canine LES, VIP receptor distribution and characterization were analyzed by radioligand binding and cross-linking experiments. RESULTS High densities of saturable VIP receptors were found (maximum bound [Bmax], 539.2 fmol/mg in the synaptosome-enriched fraction [P2] and 732.7 fmol/mg in the smooth muscle, plasma membrane-enriched fraction [Mic II]), with high affinity for 125I-VIP (dissociation constant [Kd], 1.38 nmol/L in P2 and 1.40 nmol/L in Mic II). Competition binding studies suggested the presence of two binding sites, a high-affinity (inhibitor constant [Ki1], 0.064 nmol/L) and a low-affinity (Ki2, 2.68 nmol/L) binding site in P2 membranes, but only one binding site (Ki, 1.18 nmol/L) in Mic II membranes. Guanosine triphosphate-gamma-s pretreatment eliminated high-affinity binding in P2 membranes by conversion to binding sites of lower affinity (Ki, 2.82 nmol/L). Studies with a cross-linking agent identified VIP receptors in synaptosomal and smooth muscle plasma membrane fractions; a single polypeptide of approximately 60 kilodaltons was found in each membrane. CONCLUSIONS Specific VIP receptors exist in both synaptosomal and smooth muscle plasma membrane of canine LES.