The relationship between the free cytoplasmic Ca2+concentration, Ca2+i, and neurotransmitter release was investigated in guinea pig brain synaptosomes and the neurosecretory cell line PC12. Release was induced by α -latrotoxin, which acts in both Ca2+-containing and Ca2+-free incubation media, or by the classical depolarizing agents high K+and veratridine, which require extracellular Ca2+. Two complementary approaches were used to reveal changes of Ca2+i: (i) direct measurement by a fluorescent Ca2+indicator (quin2) and (ii) study of the Ca2+-dependent phosphorylation of a protein, synapsin I, located at the cytoplasmic surface of synaptic vesicles. Depolarizing agents, when applied in Ca2+-containing medium, induced the Ca2+ito increase promptly 3- to 6-fold, drastically increased synapsin I phosphorylation, and caused stimulation of transmitter release. With α -latrotoxin, the Ca2+iincrease was delayed and occurred at a slower rate, the increase of synapsin I phosphorylation was less drastic, and the release response was much more pronounced. In Ca2+-free medium, depolarizing agents released no transmitter and had no effect on Ca2+ior synapsin I phosphorylation, whereas with α -latrotoxin these processes were dissociated: considerable stimulation of the release without apparent change of Ca2+iand synapsin I phosphorylation. We conclude that the relationship between average Ca2+iand transmitter release is not straightforward and, in particular, that the release evoked by α -latrotoxin in Ca2+-free medium is mediated by a factor(s) other than bulk redistribution of Ca2+from intracellular stores.