1. The effects of beta-bungarotoxin on acetylcholine (ACh) synthesis, tissue content and release have been studied in the rat diaphragm. A gas chromatographic mass spectrometric assay was used to measure ACh and choline. 2. Within 30 min, beta-bungarotoxin (0.14 or 1.4 micrograms/ml.) caused a significant increase in tissue ACh content. This increase was apparent prior to the final inhibition by beta-bungarotoxin of evoked (10 Hz) ACh release. 3. The toxin enhanced the incorporation of [2H4]Ch into [2H4]ACh in both resting and stimulated preparations. 4. Hemicholinium-3 blocked the rise in diaphragm ACh normally produced by beta-bungarotoxin. 5. Beta-Bungarotoxin did not directly activate choline acetyltransferase in muscle homogenates. 6. The toxin-induced rise in tissue ACh was largely absent in Ca2+-free solutions which contained either EGTA (1 mM) or SrCl2 (2 or 10 mM). 7. Non-neurotoxic phospholipases A2, fatty acids and the neurotoxic phospholipase A2, notexin, did not cause ACh accumulation in the diaphragm. 8. Beta-Bungarotoxin did not stimulate ACh synthesis in denervated muscle. 9. The extra ACh which accumulated after beta-bungarotoxin did not contribute to enhanced release by nerve impulses even when 4-aminopyridine was added to the medium. High K+ solution and black widow spider venom were also ineffective in increasing output from toxin-treated diaphragms relative to controls that had not been treated with beta-bungarotoxin. 10. Prior injection of a rat with botulinum toxin prevented the accumulation of ACh due to beta-bungarotoxin. Tubocurarine, however, did not antagonize beta-bungarotoxin. 11. These data indicate that beta-bungarotoxin has a unique capacity to inhibit ACh release and stimulate ACh synthesis in diaphragm nerve endings. The results are discussed in terms of a possible action of beta-bungarotoxin to raise the level of ionized Ca in the nerve terminal cytosol.