1 The effects of potassium depolarization and preganglionic nerve stimulation on the metabolism of [(3)H]-choline in the isolated superior sympathetic ganglion of the rat have been studied.2 When unstimulated (resting) ganglia were incubated for 10 min with a low concentration (0.1 muM) of [(3)H]-choline (high affinity uptake), approximately 75% of the accumulated radioactivity was present as [(3)H]-phosphorylcholine, 11% was [(3)H]-acetylcholine ([(3)H]-ACh) and the remainder was unchanged [(3)H]-choline.3 Depolarization of the ganglia with K (46 mM) before their incubation with [(3)H]-choline, increased [(3)H]-choline uptake by 70% and increased [(3)H]-ACh synthesis by more than 700%, so that [(3)H]-ACh represented almost 50% of the total radioactivity recovered. In contrast, the proportion of [(3)H]-phosphorylcholine fell to 36% of the total radioactivity recovered.4 The striking effect of K-depolarization on [(3)H]-ACh synthesis in ganglia occurred at a concentration of 30 mM or above, and the maximum effect was seen at 45-50 mM.5 Chronic denervation of the ganglia abolished all the effects of high-K on [(3)H]-choline metabolism. In resting ganglia, [(3)H]-ACh formation was reduced by over 80% but [(3)H]-phosphorylcholine synthesis and the level of unchanged [(3)H]-Ch were not affected by denervation.6 Exposure of the ganglia to low-Na or hemicholinium-3 (HC-3) greatly reduced [(3)H]-ACh synthesis in control resting ganglia and almost abolished the effects of high-K on [(3)H]-ACh synthesis.7 Prevention of transmitter release with high-Mg or low-Ca medium also prevented K-depolarization from stimulating [(3)H]-ACh synthesis.8 Preganglionic nerve stimulation had an effect on [(3)H]-choline metabolism similar to that of K-depolarization. Thus, at all the frequencies studied (1-30 Hz), [(3)H]-ACh synthesis was greatly increased and [(3)H]-phosphorylcholine was reduced, the maximum effects occurring at 3 Hz.9 When ganglia were incubated with a high concentration (100 muM) of [(3)H]-choline (low affinity uptake), a different pattern of metabolism was observed. Most of the radioactivity in resting ganglia was present as unchanged [(3)H]-choline (70%) with [(3)H]-phosphorylcholine and [(3)H]-ACh representing 23% and 6% of the total radioactivity respectively. K-depolarization decreased [(3)H]-choline uptake but increased the proportions of [(3)H]-phosphorylcholine and [(3)H]-ACh to 32% and 24% of the total radioactivity respectively.10 It is concluded that in unstimulated (resting) rat sympathetic ganglia most of the [(3)H]-choline transport and metabolism occurs in postsynaptic structures. However, depolarization of the presynaptic nerve terminals appears to trigger a sodium-dependent, HC-3 sensitive, high-affinity uptake process, and causes a dramatic increase in presynaptic [(3)H]-ACh synthesis together with a fall in postsynaptic [(3)H]-phosphorylcholine synthesis. These changes in choline metabolism cannot be due to the depolarization of the nerve terminals per se, because they were abolished by high-Mg or low-Ca, i.e. when transmitter release was prevented. Thus, the increase in ACh synthesis may be triggered by a fall in the intraterminal concentration of ACh or by the changes in Ca flux induced by depolarization. Our experiments do not provide evidence on these possible mechanisms.