Ca(2+) regulation of neurotransmitter release is thought to require multiple Ca(2+) sensors with distinct affinities. However, no low-affinity Ca(2+) sensor has been identified at the synapse. We now show that piccolo/aczonin, a recently described active-zone protein with C-terminal C(2)A- and C(2)B-domains, constitutes a presynaptic low-affinity Ca(2+) sensor. Ca(2+) binds to piccolo by virtue of its C(2)A-domain via an unusual mechanism that involves a large conformational change. The distinct Ca(2+)-binding properties of the piccolo C(2)A- domain are mediated by an evolutionarily conserved sequence at the bottom of the C(2)A-domain, which may fold back towards the Ca(2+)-binding sites on the top. Point mutations in this bottom sequence inactivate it, transforming low-affinity Ca(2+) binding (100-200 microM in the presence of phospholipids) into high-affinity Ca(2+) binding (12-14 microM). The unusual Ca(2+)-binding mode of the piccolo C(2)A-domain reveals that C(2)-domains are mechanistically more versatile than previously envisaged. The low Ca(2+) affinity of the piccolo C(2)A-domain suggests that piccolo could function in short-term synaptic plasticity when Ca(2+) concentrations accumulate during repetitive stimulation.