The large glutamatergic calyx of Held synapse in the auditory brainstem has become a powerful model for studying transmitter release mechanisms, but the molecular bases of presynaptic function at this synapse are not well known. Here, we have used single-cell quantitative PCR (qPCR) to study the developmental expression of all major Synaptotagmin (Syt) isoforms in putative calyx of Held-generating neurons (globular bushy cells) of the ventral cochlear nucleus. Using electrophysiological criteria and the expression of marker genes including VGluTs (vesicular glutamate transporters), Ca(2+) binding proteins, and the transcription factor Math5, we identified a subset of the recorded neurons as putative calyx of Held-generating bushy cells. At postnatal days 12-15 these neurons expressed Syt-2 and Syt-11, and also Syt-3, -4, -7 and -13 at lower levels, whereas Syt-1 and -9 were absent. Interestingly, early in development (at P3-P6), immature bushy cells expressed a larger number of Syt-isoforms, with Syt-1, Syt-5, Syt-9 and Syt-13 detected in a significantly higher percentage of neurons. Our study sheds light on the molecular properties of putative calyx of Held-generating neurons and shows the developmental regulation of the Syt-isoform expression profile in a single neuron type.