Various agents which are known to affect intracellular levels of cAMP have been assessed for their ability to induce the release of [3H]acetylcholine ([3H]ACH) from a synaptosomal preparation derived from the guinea-pig ileum myenteric plexus. 8-Bromo-cAMP increased the release of [3H]ACh above basal levels. While 8-bromo-cGMP also increased the release, this nucleotide was far less potent than 8-bromo-cAMP. Comparison of the release caused by the cyclic nucleotides to the release induced by the nicotinic agonist dimethylphenylpiperazinium (DMPP) suggested that there is some relationship, as yet undefined, between the 8-bromo-cAMP-induced and the DMPP-induced release, while no relationship was evident between the release induced by 8-bromo-cGMP and that caused by DMPP. The 8-bromo-cAMP-induced release was Ca2+-dependent. Neither adenosine, clonidine, nor oxotremorine (all of which modulate the nicotinically-induced release) affected the 8-bromo-cAMP-induced release. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine stimulated the release of [3H]ACh as did the adenylate cyclase activator forskolin. The forskolin-induced release was not affected by adenosine, clonidine or oxotremorine. The ability of the modulators to block the nicotinically-induced release but not the release caused by the cyclic nucleotides indicates that the modulation of release evoked by nicotinic activity does not occur at a step involving protein phosphorylation.