The coupling of neuronal excitation to release of chemical transmitter at synaptic endings is mediated by an electrically gated entry of Ca²⁺-ions that within a fraction of a millisecond leads to exocytotic fusion of vesicles with the presynaptic membrane. In this rapid process, members of the synaptotagmin (Syt) family of vesicular membrane proteins are likely to function as the Ca²⁺-sensing molecular trigger. The trigger mechanism employed, however, is currently under debate. Here, using high resolution dual simultaneous pre- and postsynaptic whole cell patch clamp recordings from Syt1-deficient GABAergic neurons, we report a strong potentiation of asynchronous release in the absence of synchroonous release. In addition, we show that Syt1-deficient vesicles are hypersensitive to Ca²⁺-independent osmotic stimulation of exocytosis and that, unlike in controls, chemical priming into a highly fusion competent state directly results in their spontaneous exocytosis. These findings argue strongly that an inhibitory, latch-like action by Ca²⁺-free Syt1 on SNARE-function is part of its physiological role in the mammalian nervous system, in ensuring transmitter release closely timed to the presynaptic action potential.