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1. The effects of beta-bungarotoxin on acetylcholine turnover were studied in neonatal rat diaphragms using a gas chromatographic mass spectrometric assay to measure acetylcholine and choline. 2. Injection of neonatal rats (1-15 days old) with a fixed dose of beta-bungarotoxin resulted in a shorter time to death in the older animals. 3. This increased lethal potency of beta-bungarotoxin in the older rats was correlated with an enhancement of the stimulatory effect of the toxin on acetylcholine synthesis in diaphragm in vivo and in vitro. 4. The reduced effect of beta-bungarotoxin in promoting the accumulation of acetylcholine in new-born rat diaphragms was not due to an enhancement by the toxin of acetylcholine output from these preparations. 5. These results demonstrate that the stimulatory effect of beta-bungarotoxin on acetylcholine synthesis in diaphragm is a function of the age of the rat. Thus, beta-bungarotoxin may be valuable as a probe of the maturation of presynaptic function in rat skeletal muscle.