The release of acetylcholine (ACh) from purely cholinergic Torpedo synaptosomes was monitored continuously using a chemiluminescent assay (Israël & Lesbats, 1981 a, b). Upon prolonged K+ depolarization in the presence of Ca2+, the release of ACh was transient and returned to a steady low level in about 3 min. Addition of the Ca2+ ionophore A23187 triggered the release again, suggesting that neither depletion of the transmitter store nor an inhibition of the release mechanism itself were involved in this phasic response, but rather an inactivation of the Ca2+ entry. The release response evoked by adding Ca2+ back after exposure of the synaptosomes to high K+ (70 mM) and low Ca2+ (0.57 mM) solution inactivates as a function of the duration of the pre-depolarization with a two-component time course with rapid (tau = 5.5 s) and slow phases (tau = 143 s). This response to Ca2+ addition was more strikingly reduced as the level of depolarization during pre-treatment was increased. The inactivation was found to be dose dependent with respect to the amount of Ca2+ present during the pre-depolarization period (conditioning Ca2+). Moreover, the presence of EGTA during pre-treatment with high-K+ solutions increased the response to applied Ca2+. These observations suggest that Ca2+ entry itself was responsible for this inactivation. No inactivation was found when ACh release was induced by the depolarizing agent Gramicidin D, except when external Na+ was replaced by Li+. This result indicates that part of the Ca2+ influx promoted by Gramicidin D depends on a Na+ entry, and may be mediated by the Na-Ca exchange mechanism.