Synaptotagmin-1 and -2 are known Ca(2+) sensors for fast synchronous neurotransmitter release, but the potential Ca(2+)-sensor functions of other synaptotagmins in release remain uncharacterized. We now show that besides synaptotagmin-1 and -2, only synaptotagmin-9 (also called synaptotagmin-5) mediates fast Ca(2+) triggering of release. Release induced by the three different synaptotagmin Ca(2+) sensors exhibits distinct kinetics and apparent Ca(2+) sensitivities, suggesting that the synaptotagmin isoform expressed by a neuron determines the release properties of its synapses. Conditional knockout mice producing GFP-tagged synaptotagmin-9 revealed that synaptotagmin-9 is primarily expressed in the limbic system and striatum. Acute deletion of synaptotagmin-9 in striatal neurons severely impaired fast synchronous release without changing the size of the readily-releasable vesicle pool. These data show that in mammalian brain, only synaptotagmin-1, -2, and -9 function as Ca(2+) sensors for fast release, and that these synaptotagmins are differentially expressed to confer distinct release properties onto synapses formed by defined subsets of neurons.