This study examined the possibility that membrane phospholipids might be a source of choline used for acetylcholine (ACh) synthesis. Slices of rat striatum or cerebellum were superfused with a choline-free or choline-containing (10, 20 or 40 microM) physiological solution with eserine, for alternating 20 min periods of rest or electrical stimulation. Superfusion media were assayed for choline and ACh, and slice samples taken before and after stimulation were assayed for choline, ACh, various phospholipids, protein and DNA. The striatal slices were able to sustain the stimulation-induced release of ACh, releasing a total of about 3 times their initial ACh contents during the 8 periods of stimulation and rest. During these 8 cycles, 885 pmol/micrograms DNA free choline was released from the slices into the medium, an amount about 45-fold higher than the initial or final free choline levels in the slices. Although repeated stimulation of the striatal slices failed to affect tissue levels of free choline or of ACh, this treatment did cause significant, dose-related (i.e., number of stimulation periods) stoichiometric decreases in tissue levels of phosphatidylcholine (PC) and of the other major phospholipids; tissue protein levels also declined significantly. Addition of exogenous choline to the superfusion medium produced dose-related increases in resting and evoked ACh release. The choline also fully protected the striatal slices from phospholipid depletion for as many as 6 stimulation periods. Cerebellar slices liberated large amounts of free choline into the medium but did not release measurable quantities of ACh; their phospholipid and protein levels did not decline with electrical stimulation. These data show that membrane phospholipids constitute a reservoir of free choline that can be used for ACh synthesis. When free choline is in short supply, ACh synthesis and release are sustained at the expense of this reservoir. The consequent reduction in membrane PC apparently is associated with a depletion of cellular membrane. The use of free choline by cholinergic neurons for two purposes, the syntheses of both ACh and membrane phospholipids, may thus impart vulnerability to them in situations where the supply of free choline is less than that needed for acetylation.