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1. Acetylcholine (ACh) released from mouse diaphragm was gel filtrated and estimated by bio-assay and compared with electrophysiologically measured quantal release, expressed either as frequency of miniature end-plate potentials or quantum content of end-plate potentials. 2. Activation of Na+-K+-dependent membrane ATPase (membrane ATPase) in Na+-loaded muscles lowered the total amount of ACh released at rest to one tenth of the control value, but quantal release remained unchanged. 3. Inhibition of membrane ATPase by 2 X 10(-5) M-ouabain or by K-free solution led to an increase in total release and to a delayed progressive increase in quantal release. When Ca2+ was removed only the total release was enhanced. 4. Depolarization of the diaphragm by 8, 11 and 14 mM-K increased both total and quantal release only in the presence of Ca2+ in the perfusion medium. When Ca2+ was removed, no significant increase in release was observed. 5. The total and quantal release in response to 2 Hz stimulation of the preparation was increased 1.4 and 45 times, respectively. It is concluded that the total amount of ACh released at rest consists of two fractions, quantal and non-quantal, the former representing about 1% of the total release.