Previous experiments showed that exposure of sympathetic ganglia to exogenous adenosine increased acetylcholine (ACh) content and its subsequent release. This effect was not mediated through extracellular adenosine receptors, but at an intracellular site following its uptake through nitrobenzylthioinosine (NBTI)-resistant nucleoside transporters. We postulated that endogenous adenosine may play a role in modulating synaptic transmission in the superior cervical ganglion. The present study tested whether adenosine is involved in the activation of ACh synthesis that occurs during a rest period following prolonged presynaptic tetanic activity. Conditioning of ganglia with high-frequency stimulation (15 Hz) for 45 min followed by a 15 min rest increased their ACh content by 45%. The appearance of this “rebound ACh” showed sensitivity to nucleoside transport inhibitors; it was prevented by dipyridamole, but not by NBTI or meclonazepam, and it was reduced in the presence of RO 11–3624, suggesting an involvement of NBTI-resistant transporters. The effect of dipyridamole was specific for the synthesis of rebound ACh in that it did not inhibit ACh release or ACh synthesis during stimulation. The inhibitory action of dipyridamole on the synthesis of rebound ACh was not evident if it was present only during the tetanic stimulation but it was if dipyridamole was present during the rest period following it, suggesting that adenosine's presence after tetanic stimulation is of importance. This conclusion was strengthened by experiments showing that the presence of cyclopentyltheophylline, an antagonist at inhibitory adenosine receptors, increased ACh output evoked by test stimulation immediately following tetanic activity, as if endogenous adenosine was available at that time to activate the adenosine receptors that inhibit transmitter release. ACh release from conditioned ganglia was 44% greater than that from the controls. However, the rebound ACh was not mobilized in the presence of 2-(4-phenylpiperidino)cyclohexanol (vesamicol), a vesicular ACh transporter inhibitor. These results suggest that endogenous adenosine released after tetanic stimulation activates ACh synthesis, which results in an increase of ganglionic ACh that is available for subsequent mobilization and release.