A proline scan at positions 2 and 3 of the opioid peptide dynorphin A(1-11)-NH(2) led to the discovery of the analogue [Pro(3)]Dyn A(1-11)-NH(2). This analogue possesses high affinity and selectivity for the kappa opioid receptor (K(i)(kappa) = 2.7 nM, K(i) ratio kappa/micro/delta = 1/2110/3260). The gain in selectivity is achieved through an overall reduction of opioid receptor affinity which is most pronounced at micro and delta receptors. The Pro(3) analogue exhibits antagonist properties. Despite its high kappa affinity, [Pro(3)]Dyn A(1-11)-NH(2) is a relatively weak antagonist in both the [(35)S]GTPgammaS assay (IC(50) = 380 nM) and the guinea pig ileum assay (K(e) = 244 nM). Discrepancies between GPI and binding assay have often been ascribed to differential kappa receptor subtypes prevailing in central vs peripheral neurons. Since the [(35)S]GTPgammaS assay uses the same membrane preparations as the binding assay, differential kappa subtypes can be ruled out as an explanation in this case, and the observed behavior rather seems to reflect an intrinsic property of the ligand.