Mitochondria located within neuronal presynaptic terminals have been shown to play important roles in the release of chemical neurotransmitters. In the present study, a genetic screen for synaptic transmission mutants of Drosophila has identified the first mutation in a Drosophila homolog of the mitochondrial protein P32. Although P32 is highly conserved and has been studied extensively, its physiological role in mitochondria remains unknown and it has not previously been implicated in neural function. The Drosophila P32 mutant, referred to as dp32(EC1), exhibited a temperature-sensitive (TS) paralytic behavioral phenotype. Moreover, electrophysiological analysis at adult neuromuscular synapses revealed a TS reduction in the amplitude of excitatory postsynaptic currents (EPSC) and indicated that dP32 functions in neurotransmitter release. These studies are the first to address P32 function in Drosophila and expand our knowledge of mitochondrial proteins contributing to synaptic transmission.