1. The effect of the opioid peptides [Met5]enkephalin-Arg6-Phe7 (MEAP) and [Met5]enkephalin-Arg6-Gly7-Leu8 (MEAGL) were compared with those of [Leu5]enkephalin and [D-Ala2,Met5]enkephalinamide (DAME) on cholinergic neurotransmission in the rabbit isolated atria. 2. Rabbit isolated atria had a resting rate of 190 beats/min. In the presence of the beta-adrenoceptor antagonist propranolol (0.3 mumol/l), atria responded to electrical field stimulation with a cholinergically mediated negative chronotropic response. The opioid peptides had no effect on the resting rate, but inhibited the negative chronotropic response to field stimulation. The IC50 values for inhibiting the cholinergic responses were 1.4 mumol/l for [Leu5]enkephalin (LE), 1.4 mumol/l for MEAP, 1.3 mumol/l for MEAGL and 0.2 mumol/l for DAME. Responses of a similar magnitude to exogenous acetylcholine were unaffected. 3. Thus, MEAP, MEAGL and LE had similar potencies but DAME was about seven times more potent in inhibiting cholinergic neurotransmission in the rabbit isolated atria. The site of inhibition appears to be prejunctional.