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The mode of action of cholecystokinin (CCK), an octapeptide fragment of CCK, caerulein, and pentagastrin on isolated guinea pig ileal muscle was investigated and their relative potencies determined. On a molar basis, octapeptide-CCK and caerulein were about twice as potent as CCK, whereas pentagastrin and Urecholine were respectively 23 and 46 times less potent. Scopolamine (6.84 × 10−4 M) inhibited the response to all four peptides relatively less than the response to Urecholine. Tetrodotoxin (0.1 μg/ml) inhibited the response to all peptides by 91%. It was concluded that the effect of these peptides on ileal muscle was largely mediated by cholinergic mechanisms. It could not be ascertained, however, whether the effect resulted solely from release of acetylcholine by nerve endings or from the participation of acetylcholine mainly as a synergist of the peptides' action on smooth muscle fibers.