Temperature-sensitive mutations of the choline acetyltransferase (Cha) gene, which lead to reduced choline acetyltransferase (ChAT) activity and acetylcholine (ACh) levels, have been used in an attempt to identify the neurotransmitter at a chemical synapse in the giant fiber pathway (GFP) of Drosophila melanogaster. Prolonged incubation of adult mutant flies at non-permissive temperatures blocked the response of this pathway to brain stimulation, whereas shorter incubation times disturbed various parameters of the normal response. Even at permissive temperature subnormal responses were still evident. These defects in the giant fiber pathway's function suggest a specific cholinergic synapse within the pathway, the first synapse of this type implicated in Drosophila. When the function of this synapse was experimentally stressed, disruptions of GFP function paralleling decreased ChAT activity began to appear at enzyme levels estimated to be approximately 80% of wild-type.