1. Homogenate binding studies and receptor autoradiography have been used to examine the binding characteristics and localization of propranolol-resistant (-)-[125I]-cyanopindolol (CYP) binding sites in rat ileum. 2. Saturation studies with (-)-[125I]-CYP and homogenates of rat ileum identified a site with pKD 8.89 +/- 0.08 and Bmax = 50.3 +/- 4.1 fmol mg-1 protein (n = 6). Both beta 1- and beta 2-adrenoceptors (AR) were not detected in these preparations. 3. (-)-Isoprenaline infusion (400 micrograms kg-1 h-1) for 14 days caused no significant change in the density of (-)-[125I]-CYP binding which was 48.9 +/- 12.8 and 40.6 +/- 12.3 fmol mg-1 protein in control and isoprenaline-treated animals respectively (n = 6) (P = 0.97). 4. Competition for (-)-[125I]-CYP binding in the presence of 0.1 microM (-)-propranolol gave affinity values for CYP, tertatolol, alprenolol, ICI 118551 and CGP 20712A that correspond to known affinities at atypical beta-ARs. Stereoselectivity ratios for tertatolol and alprenolol were low. 5. Autoradiographic localization of propranolol resistant (-)-[125I]-CYP binding showed sites associated with the mucosa and to a lesser extent to the muscularis. A small population of beta 2-ARs were detected located predominantly in the longitudinal and circular smooth muscle layers. 6. This study identifies an (-)-[125I]-CYP binding site in rat ileum that is resistant to blockade by propranolol (0.1 microM), is located predominantly in the mucosa, shows resistance to downregulation by isoprenaline and has binding characteristics of the atypical beta-AR.