We studied inactivation of Ca²⁺-induced Ca²⁺ release (CICR) via ryanodine receptors (RyRs) in bullfrog sympathetic neurons. The rate of rise in [Ca²⁺]_i due to CICR evoked by a depolarizing pulse decreased markedly within 10-20 ms to a much slower rate despite persistent Ca²⁺ entry and little depletion of Ca²⁺ stores. The Ca²⁺ entry elicited by the subsequent pulse within 50 ms, during which the [Ca²⁺]_i level remained unchanged, did not generate a distinct [Ca²⁺]_i rise. This mode of [Ca²⁺]_i rise was unaffected by a mitochondrial uncoupler, carbonyl cyanide p-trifluromethoxy-phenylhydrazone (FCCP, 1 µM). Paired pulses of varying interval and duration revealed that recovery from inactivation became distinct 50 ms after depolarization and depended on [Ca²⁺]_i. The inactivation was prevented by BAPTA (100 µM) but not by EGTA (10 mM), whereas the activation was less affected by BAPTA. When CICR was partially activated, some of the non-activated RyRs were also inactivated directly. Thus the inactivation in these neurons is induced by Ca²⁺ binding to the high affinity regulatory sites residing very close to Ca²⁺ channels and/or RyRs, although the sites for activation are located much closer to those Ca²⁺ sources. The rate of [Ca²⁺]_i decay after the pulse decreased with increasing pulse duration longer than 10 ms, and this was abolished by BAPTA. Thus some mechanism counteracting Ca²⁺ clearance is induced after full inactivation and potentiated during the pulse. Possible models for RyR inactivation were proposed and the roles of inactivation in Ca²⁺ signalling were discussed.