We incorporated bromoacetylcholine (an inhibitor of choline acetyltransferase), acetylcholinesterase, or both into liposomes made of phosphatidylcholine. Frog sartorius muscles were exposed to these liposomes for 30-60 min. The liposome treatment itself did not decrease the size of the quanta compared to untreated controls. Then the preparations were exposed for 10-20 min to a hypertonic solution, which increases the rate of spontaneous quantal release and elicits an increase in the amount of acetylcholine released per quantum. Following the hypertonic treatment the quanta were significantly smaller in the liposome-treated preparations. Most of the difference occurred because in the preparations not exposed to the liposomes quantal size increased following the hypertonic treatment. This increase is thought to be due to the incorporation of more acetylcholine into each quantum. Our conclusions are that the treatments decreased the acetylcholine concentration in the cytoplasm, and that the increase in size occurs because additional acetylcholine is added to the vesicles containing the quanta from the cytoplasm.