The present experiments tested whether acetate plays a role in the provision of acetyl-CoA for acetylcholine synthesis in the cat's superior cervical ganglion. Labeled acetylcholine was identified in extracts of ganglia that had been perfused for 20 min with Krebs solution containing choline (10(-5) M) and [3H], [1-14C], or [2-14C]acetate (10(-3) M); perfusion for 60 min or with [3H]acetate (10(-2) M) increased the labeling. The acetylcholine synthesized from acetate was available for release by a Ca2+-dependent mechanism during subsequent periods of preganglionic nerve stimulation. When ganglia were stimulated via their preganglionic nerves or by exposure to 46 mM K+, the labeling of acetylcholine from [3H]acetate was reduced when compared with resting ganglia. The reduced synthesis of acetylcholine from acetate during stimulation was not due to acetate recapture, shunting of acetate into lipid synthesis, or the transmitter release process itself. In ganglia perfused with [2-14C]glucose, the amount of labeled acetylcholine formed was clearly enhanced during stimulation. An increase in acetylcholine labeling from [3H]acetate was shown during a 15-min resting period following a 60-min period of preganglionic nerve stimulation (20 Hz). It is concluded that acetate is not the main physiological acetyl precursor for acetylcholine synthesis in this sympathetic ganglion, and that during preganglionic nerve stimulation there is enhanced delivery of acetyl-CoA to choline acetyltransferase from a source other than acetate.