1 The effects of opioids on the sacral parasympathetic outflow to cat distal colon were studied in vitro using muscle strips orientated in the axis of the longitudinal muscle layer, with pelvic nerves attached. Electrical stimulation of the pelvic nerves evoked contractions that were blocked by atropine (1 X 10(-6) M) and tetrodotoxin (3 X 10(-7) M). 2 [D-Pen2, D-Pen5]enkephalin and [Met5]- and [Leu5]enkephalin caused concentration-dependent, reversible inhibition of pelvic nerve-evoked contractions, with IC50 values of 8.3 X 10(-10) M, 2.2 X 10(-9) and 2.1 X 10(-9) M respectively. 3 Morphine (1 X 10(-7)-1 X 10(-5) M) and [D-Ala2, MePhe4, Gly-ol5]enkephalin (1 X 10(-8)-1 X 10(-6) M) and U-50,488H (1 X 10(-8)-10(-6) M) were much less potent as inhibitors than [Met5]- or [Leu5]enkephalin. 4 Naloxone (1 X 10(-7) M), an antagonist at each of the three opioid receptor types, antagonized the effects of both [Met5]enkephalin and morphine. However, ICI 174,864, a specific delta-opioid receptor antagonist, antagonised the effects of [Met5]enkephalin only. 5 The inhibitory actions of [Met5]enkephalin were inversely related to frequency of pelvic nerve stimulation. Also, [Met5]enkephalin at a concentration (3 X 10(-9) M) which produced a large inhibition of neurogenic contractions, had no effect on contractions to exogenous acetylcholine. These results suggest a prejunctional site for inhibitory opioid receptors. 6 In summary, prejunctional inhibitory delta-opioid receptors are present on the sacral parasympathetic outflow to cat distal colon; kappa- and/or mu-opioid receptors may also be present, but appear to be of lesser importance.