Abstract The choice of opiate receptor subtype by α- and β-neo-endorphin was studied in isolated preparations. Neo-endorphins had significant inhibitory actions in the electrically evoked contractions of guinea-pig ileum, mouse vas deferens and rabbit ileum as well as on the rabbit vas deferens which had been shown to contain κ-receptors exclusively. Mr 2266, a relatively specific κ-receptor antagonist, was more effective than naloxone, a relatively specific μ-receptor antagonist, to antagonize the agonist actions of neo-endorphins in either the guinea-pig and rabbit ileum or in the rabbit vas deferens. By contrast, in the mouse vas deferens, the effectiveness of Mr 2266 to antagonize the agonist actions of neo-endorphins was low and similar to that of naloxone. The potencies of neo-endorphins relative to that of ethylketocyclazocine, a representative κ-receptor agonist, in the guinea-pig ileum were similar to those in the rabbit ileum but were significantly different from those in the mouse vas deferens. The data indicate that neo-endorphins act as κ-receptor agonists in either the guinea-pig and rabbit ileum or in the rabbit vas deferens while in the mouse vas deferens they act on opiate receptor subtypes other than κ- and μ-receptors.