Neurotransmission at chemical synapses involves regulated exocytosis of neurotransmitter from the presynaptic terminal. Neurotransmitter release is thought to be triggered by calcium influx through specific classes of voltage-gated calcium channels. Here we report genetic and functional analysis implicating a specific calcium channel gene product in neurotransmitter release. We have isolated a temperature-sensitive paralytic allele of the Drosophila calcium channel alpha1 subunit gene, cacophony (cac). This mutant, referred to as cac(TS2), allows functional analysis of synaptic transmission after acute perturbation of a specific alpha1 subunit. Electrophysiological analysis at neuromuscular synapses revealed that neurotransmitter release in cac(TS2) is markedly reduced at elevated temperatures, indicating that cac encodes a primary alpha1 subunit functioning in synaptic transmission. These observations further define the molecular basis of voltage-gated calcium entry at synapses and provide a new starting point for further genetic analysis of synaptic mechanisms.