Autoradiographic binding studies using [125I]S(-)-zacopride (0.1 nM) identified non-5-HT3 specific binding sites (defined by 5-hydroxytryptamine (5-HT), 1.0 microM) in the rat duodenum and ileum and some other peripheral tissues (adrenal gland, liver, stomach, kidney and spleen). In the rat duodenum and ileum, saturation studies with [125I]S(-)-zacopride indicated that the specific binding was saturable and of high affinity to an apparently homogenous population of binding sites (duodenum Bmax = 1.88 fmol/mg, Kd = 0.078 nM; ileum Bmax = 1.60 fmol/mg, Kd = 0.071 nM). Competition studies with slices of either duodenum or ileum indicated that the pharmacology of the [125I]S(-)-zacopride recognition site in both tissues was comparable but differed from all 5-HT receptors and uptake sites reported to date. However, the [125I]S(-)-zacopride recognition site displayed some pharmacological and regional similarity to the 5-HT1P recognition site: The sensitivity of the [125I]S(-)-zacopride binding in the duodenum and ileum to GTP indicates that the radiolabelled recognition site may represent a functional G-protein coupled receptor.