This study investigated the effects of BIIE0246, a novel neuropeptide Y (NPY) Y2receptor antagonist, on the inhibition of cholinergic neuroeffector transmission in rat heart and guinea‐pig trachea and purinergic neuroeffector transmission in guinea‐pig vas deferens produced by the NPY Y2receptor agonist, N‐acetyl[Leu28,31] NPY 24‐36.In pentobarbitone anaesthetized rats, supramaximal stimulation every 30 s, of the vagus nerve innervating the heart, increased pulse interval by approximately 100 ms. This response was attenuated by intravenous administration of N‐acetyl[Leu28,31] NPY 24‐36 (10 nmol kg−1).Transmural stimulation of segments of guinea‐pig trachea at 1 min intervals with 5 s trains of stimuli at 0.5, 5, 10, 20 and 40 Hz evoked contractions which were reduced in force by N‐acetyl[Leu28,31] NPY 24‐36 (2 μM).In guinea‐pig vasa deferentia, the amplitude of excitatory junction potentials evoked by trains of 20 stimuli at 1 Hz was reduced in the presence of N‐acetyl[Leu28,31] NPY 24‐36 (1 μM).In all preparations BIIE0246 attenuated the inhibitory effect of N‐acetyl[Leu28,31] NPY 24‐36 but had no effect when applied alone.The findings support the view that the nerve terminals of postganglionic parasympathetic and sympathetic neurones possess neuropeptide Y Y2receptors which, when activated, reduce neurotransmitter release.