Frog sartorius muscles were homogenized under various conditions which allowed, by means of mass spectrometry, the measurement of total ACh, and different ACh compartments in the tissue: 'bound', 'free-1' and 'free-2' ACh. Bound ACh presumably corresponded to the vesicular compartment, and the free-1 and free-2 fractions to the cytoplasmic compartments of ACh. Stimulation of ACh release by La3+ ions for 60 min caused a decrease of both bound and free-2 ACh, but at 20 min bound ACh was reduced much more than free-2 ACh. Stimulation of ACh release by isotonic potassium propionate (KPr) solution for only 5 min caused a decrease of bound ACh, in contrast to free-1 and free-2 ACh which were not significantly changed. When muscles after 5 min stimulation in KPr were allowed to recover in normal Ringer, free-1 ACh did not change, but free-2 and bound ACh increased; after 180 min in Ringer bound ACh had recovered to control values. When ACh synthesis was prevented by hemicholinium-3 during recovery of the muscles in Ringer, bound ACh increased at the expense of free-2 ACh. In deuterium labeling experiments, in which the Ringer contained choline-d9, much more ACh-d9 was formed in stimulated than in unstimulated muscles. It appeared that almost all newly formed ACh was ACh-d9, since no significant synthesis of unlabeled ACh (ACh-d0) took place. Yet again, the amount of bound ACh-d0 significantly increased, apparently at the expense of preformed free-2 ACh-d0.(ABSTRACT TRUNCATED AT 250 WORDS)