The turnover of acetylcholine (ACh) in rat brain synaptosomes and its compartmentation in the labile bound and stable bound pools were investigated. The P(2) fraction from rat brain was subjected to three sequential incubations, each terminated by centrifugation followed by determination of ACh concentrations by gas chromatography-mass spectrometry (GCMS): (1) Depletion phase: Incubation of synaptosomes at 37 percent C for 10 min in Na+ -free buffer containing 35 mM-KCl reduced the content of both labile bound and stable bound ACh by 40%. (2) Synthesis phase: incubation at 37 percent C with 2 micrometer-[(2)H(4)]choline resulted in accumulation of labeled and unlabeled ACh in both compartments. Addition of an anticholinesterase had little effect on stable bound ACh but greatly increased the content of labile bound ACh. This excess accumulated ACh was probably due to inhibition of intracellular acetylcholinesterase (AChE), because negligible uptake of ACh from the medium was observed. The effects on ACh synthesis of altered cation concentrations and metabolic inhibitors were examined. (3) Release phase: The tissue was incubated in the presence of 35 mM-KCl, 40 micrometer-paraoxon, and 20 micrometer-hemicholinium-3 (HC-3) (to inhibit further synthesis of ACh). Measurements of the compartmental localization of ACh at several time points indicated that ACh was being released from the labile bound fraction. In support of this conclusion, 20 mM-Mg2+ reduced ACh release and increased the labile bound ACh concentration.