Voltage-dependent calcium channels regulate many aspects of neuronal biology, including synaptic transmission. In addition to their alpha1 subunit, which encodes the essential voltage gate and selective pore, calcium channels also contain auxiliary alpha2delta, beta, and gamma subunits. Despite progress in understanding the biophysical properties of calcium channels, the in vivo functions of these auxiliary subunits remain unclear. We have isolated mutations in the gene encoding an alpha2delta calcium channel subunit (d alpha2delta-3) using a forward genetic screen in Drosophila. Null mutations in this gene are embryonic lethal and can be rescued by expression in the nervous system, demonstrating that the essential function of this subunit is neuronal. The photoreceptor phenotype of d alpha2delta-3 mutants resembles that of the calcium channel alpha1 mutant cacophony (cac), suggesting shared functions. We have examined in detail genotypes that survive to the third-instar stage. Electrophysiological recordings demonstrate that synaptic transmission is severely impaired in these mutants. Thus the alpha2delta calcium channel subunit is critical for calcium-dependent synaptic function. As such, this Drosophila isoform is the likely partner to the presynaptic calcium channel alpha1 subunit encoded by the cac locus. Consistent with this hypothesis, cacGFP fluorescence at the neuromuscular junction is reduced in d alpha2delta-3 mutants. This is the first characterization of an alpha2delta-3 mutant in any organism and indicates a necessary role for alpha2delta-3 in presynaptic vesicle release and calcium channel expression at active zones.