The release of 3H-acetylcholine (ACh) from longitudinal muscle strips of guinea pig ileum, which were previously incubated with 3H-choline, was measured by scintillation spectrometry. The release of ACh evoked by electrical field stimulation was inhibited in the following ways: stimulating muscarinic receptors directly with oxotremorine or indirectly with eserine by increasing ACh concentration in the surrounding axon terminals or stimulating adenosine receptors by increasing the biophase concentration of adenosine with dipyridamole. The muscarinic antagonist atropine and the adenosine receptor antagonist theophylline enhanced ACh release. Atropine prevented the effect of eserine and oxotremorine on ACh release and theophylline counteracted the effect of dipyridamole. When the release of ACh was under the inhibitory effect of muscarinic receptor stimulation theophylline did not increase ACh release. Under these conditions atropine caused an extremely high increase in the release of ACh, which was not further enhanced by theophylline. When the extracellular level of adenosine was increased by dipyridamole, eserine, atropine or eserine and atropine together, they were unable to change the release of ACh, while theophylline increased release of ACh. Therefore, it is concluded that the muscarinic receptor mediated inhibition of ACh release is not due to previously released adenosine. Thus, adenosine and muscarinic feedback systems seem to be independent and each cholinergic nerve ending contains both adenosine and muscarinic receptors.