Prostaglandin E2 (PGE2) induced a dose-dependent increase in tone of the circular muscles of guinea pig ileum in vitro. These actions of PGE2 were deleted in the cold-stored preparations and blocked by tetrodotoxin. Atropine reduced the effects of PGE2 and physostigmine potentiated the PGE2-induced contractions. The release of acetylcholine (ACh) by PGE2 was responsible for initiating this contraction. The effect of PGE2 was compared with that of an electrical stimulation which also initiated a non-receptor-mediated release of ACh. Hexamethonium abolished the effect of PGE2 but did not influence the actions of the electrical stimulations. Synaptosomal fractions of the circular muscles were prepared to study the release of [14C]ACh. However, PGE2 failed to evoke a marked increase in the efflux of radioactivity, even at the maximal concentration. Damage and/or removal of the myenteric plexus may be responsible for this result because electrical stimulations that exert a powerful spasmogenic effect on longitudinal muscles also induced an insensitive response. Alloxan and ethacrynic acid, inhibitors of adenylate cyclase, reduced the activity of PGE2 at a concentration insufficient to modify either the actions of ACh or the electrical stimulations. 3-Isobutyl-1-methylxanthine (IBMX) potentiated the responses to PGE2 at a dose sufficient to block the activity of phosphodiesterase (PDE). Imidazole, a stimulator of PDE, decreased the actions of PGE2 in a dose-dependent manner. IBMX, like imidazole, failed to modify the activities of both ACh and the electrical stimulations. These results indicate that PGE2 may function as a releaser of ACh in a cyclic AMP-dependent manner in the circular muscles of guinea pig ileum.