Developmental refinement of synaptic transmission can occur via changes in several pre- and postsynaptic factors, but it has been unknown whether the intrinsic Ca2+ sensitivity of vesicle fusion in the nerve terminal can be regulated during development. Using the calyx of Held, a giant synapse in the auditory pathway, we studied the presynaptic mechanisms underlying the developmental regulation of Ca2+-secretion coupling, comparing a time period before, and shortly after the onset of hearing in rats. We found an similar to 2-fold leftward shift in the relationship between EPSC amplitude and presynaptic Ca2+ current charge (Q(Ca)), indicating that brief presynaptic Ca2+ currents become significantly more efficient in driving release. Using a Ca2+ tail current protocol, we also found that the high cooperativity between EPSC amplitude and Q(Ca) was slightly reduced with development. In contrast, in presynaptic Ca2+ uncaging experiments, the intrinsic Ca2+ cooperativity of vesicle fusion was identical, and the intrinsic Ca2+ sensitivity was slightly reduced with development. This indicates that the significantly enhanced release efficiency of brief Ca2+ currents must be caused by a tighter co-localization of Ca2+ channels and readily releasable vesicles, but not by changes in the intrinsic properties of Ca2+-dependent release. Using the parameters of the intrinsic Ca2+ sensitivity measured at each developmental stage, we estimate that during a presynaptic action potential (AP), a given readily releasable vesicle experiences an about 1.3-fold higher 'local' intracellular Ca2+ concentration ([Ca2+](i)) signal with development. Thus, the data indicate a tightening in the Ca2+ channel-vesicle co-localization during development, without a major change in the intrinsic Ca2+ sensitivity of vesicle fusion.