1 We measured the effects of agents, which are thought to alter the Ca2+ concentration in cells, on tissue and released acetylcholine and choline of rat diaphragm using a gas chromatographic mass spectrometric assay for acetylcholine and choline. 2 Lanthanum and ouabain increased the resting output of acetylcholine, while 4-aminopyridine enhanced the evoked output of acetylcholine. 3 Guanidine first increased and then depressed acetylcholine release. 4 Theophylline, dinitrophenol and the ionophore, A-23187, had no measurable effect on acetylcholine release and tetraethylammonium inhibited the release of acetylcholine. 5 Dinitrophenol caused a highly significant increase of the tissue and released choline. 6 None of these agents increased the tissue acetylcholine content. Tetraethylammonium caused a large decline of tissue acetylcholine while ouabain and guanidine caused smaller reductions of acetylcholine in diaphragm. 7 These results are discussed in relation to the hypothesis that intraterminal Ca2+ activity regulates the synthesis of acetylcholine in diaphragm nerve endings.