Neuropeptide Y (NPY) is widely distributed in the central and peripheral nervous systems where it serves neuromodulator and neurotransmitter functions. NPY is contained within intrinsic nerves of the small intestine and can be demonstrated to inhibit intestinal secretion when added to the serosal side of intestine mucosa mounted in Ussing chambers. When injected centrally it has potent effects on food intake, blood pressure, sexual activity and circadian rhythms. Using NPY radiolabeled with iodogen, lactoperoxidase, or the Bolton-Hunter reagent, we have localized high-affinity NPY receptors on brain membranes and on the serosal laterobasal membranes of the rat intestinal epithelial cell. We have demonstrated that enzymatic degradation may limit the ability to demonstrate NPY binding to brush border membranes. In other experiments NPY was cross-linked to its receptors in brain and intestine using disuccinimido suberate and the resulting complexes analyzed on SDS polyacrylamide gel electrophoresis followed by radioautography. We identified two main NPY receptor species in the intestine with molecular sizes of 52-59 kDa and 37-39 kDa. The 37-39 kDa species may possess a disulfide bond which gives the receptor a fixed conformation, or it may be composed of two subunits (37-39 kDa and approximately 5 kDa subunits). This conclusion is based on the different migration of the smaller band in the presence of the reducing agent, dithiothreitol. The intestinal NPY receptor exhibits differences from the rat brain receptor previously characterized by us using similar techniques. The brain receptor has a molecular weight of approximately 58 kDa with a smaller species of about 35 kDa which shows no differences in migration after exposure to dithiothreitol. The localization of NPY receptors on laterobasal membranes and brain membranes is consistent with previous anatomic and physiologic findings. The different characteristics of each receptor type provides physical evidence of receptor heterogeneity. However, it is possible that the greater enzymatic degradation observed in intestinal membranes might explain the differences in receptor sizes in the two organs.