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The neuropeptides somatostatin and galanin are present in the iris and may modulate pupil diameter. We examined the effects of somatostatin and galanin on isolated rabbit iris dilator and sphincter smooth muscles that were mounted in an organ bath. An isometric transducer recorded changes in tension in response to electric field stimulation (100 Hz, 0.3 m sec in duration, 10 V in strength) delivered by a pair of platinum plate electrodes. The dilator muscle response to field stimulation was not changed by either peptide, even at the highest concentrations examined. The sphincter muscle response consisted of two components: a fast component mediated by acetylcholine and slow component mediated by substance P. Both somatostatin and galanin attenuated the cholinergic component in a dose-dependent manner (from 0.3 nm to 0.1 μm) but had no effect on responses mediated by substance P. Galanin was more effective (attenuation of 43% at 0.1 μm) compared with somatostatin (attenuation of 16% at 0.1 μm) in reducing the cholinergic response. Neither peptide affected the contraction induced by acetylcholine (1 mm). Therefore both peptides inhibited cholinergic transmission in the sphincter muscle, although the degree of inhibition by each was different. We conclude that somatostatin and/or galanin may induce mydriasis by attenuating cholinergic neurotransmitter release.