Synaptic transmission requires spatial and temporal coordination of a specific sequence of events. The trigger for synaptic vesicle exocytosis is Ca(2)(+) entry into presynaptic terminals, leading to neurotransmitter release at highly specialized sites known as active zones. Ca(2)(+) channel proximity to exocytotic proteins and vesicle clusters at active zones have been inferred from biochemical, histological and ultrastructural data, but direct evidence about functional relationships between these elements in central synapses is absent. We have utilized the lamprey giant reticulospinal synapse to characterize functional colocalization of known synaptic markers in the presynaptic terminal, as well as their reliability during repeated activation. Recycling vesicle clusters, surrounding actin filaments, and physiologically relevant Ca(2)(+) influx all show identical morphological distribution. Ca(2)(+) influx is mediated by clusters of Ca(2)(+) channels that colocalize with the vesicle clusters, defined by imaged sites of vesicle recycling and actin localization. Synaptic transmission is inhibited by block of actin depolymerization, but Ca(2)(+) signalling is unaffected. Functional Ca(2)(+) channels are localized to presynaptic clusters, and Ca(2)(+) transients at these sites account for neurotransmitter release based on their spatial and temporal profiles. Ca(2)(+) transients evoked by single axonal action potentials are mediated solely by voltage-operated Ca(2)(+) channel activation, and slower Ca(2)(+) rises observed throughout the axon result from Ca(2)(+) diffusion from the synaptic regions. We conclude that at lamprey giant reticulospinal synapses, Ca(2)(+) channels and release sites colocalize, creating a close spatial relationship between active zones and Ca(2)(+) entry sites, which is necessary for site-specific, Ca(2)(+)-dependent secretion.