Ca²⁺-triggered synchronous neurotransmitter release is well described, but asynchronous release—in fact, its very existence—remains enigmatic. Here we report a quantitative description of asynchronous neurotransmitter release in calyx-of-Held synapses. We show that deletion of synaptotagmin 2 (Syt2) in mice selectively abolishes synchronous release, allowing us to study pure asynchronous release in isolation. Using photolysis experiments of caged Ca²⁺, we demonstrate that asynchronous release displays a Ca²⁺ cooperativity of ∼2 with a Ca²⁺ affinity of ∼44 μM, in contrast to synchronous release, which exhibits a Ca²⁺ cooperativity of ∼5 with a Ca²⁺ affinity of ∼38 μM. Our results reveal that release triggered in wild-type synapses at low Ca²⁺ concentrations is physiologically asynchronous, and that asynchronous release completely empties the readily releasable pool of vesicles during sustained elevations of Ca²⁺. We propose a dual-Ca²⁺-sensor model of release that quantitatively describes the contributions of synchronous and asynchronous release under conditions of different presynaptic Ca²⁺ dynamics.