The sluggish-A (slg A) gene of Drosophila melanogaster has been shown to encode for the enzyme proline oxidase, a mitochondrial enzyme which catalyzes the first step in the conversion of L-proline to L-glutamate. The slg A transcript is expressed in both larval and adult Drosophila melanogaster. Mutations in this gene lead to reduced proline oxidase activity and an elevation of free proline levels. Adult mutant flies show a striking reduction of motor activity. Since proline oxidase may contribute to the supply of the neurotransmitter glutamate in the nervous system, a reduction in proline oxidase activity could reduce neural glutamate pools and affect synaptic transmission in neurons utilizing glutamate as a transmitter, including peripheral motor neurons. We tested the hypothesis that glutamate, and synaptic transmission mediated by glutamate, are reduced at synapses of glutamatergic motor neurons in slg A mutants. Levels of glutamate and proline in different cell compartments, and functional properties of synaptic transmission were compared in slg A and control specimens. Proline is elevated in muscle cells of slg A mutants, indicating that the slg A gene regulates tissue proline levels. In nerve terminal varicosities, proline levels were low in both mutants and controls. Glutamate levels in nerve terminal varicosities of slg A mutants and controls were similar. In addition, we found that glutamatergic synaptic transmission at individual nerve endings and at the whole-cell level was similar in slg A mutants and controls. Thus, proline oxidase does not play a major role in generating neuronal glutamate pools at the Drosophila larval neuromuscular junction, and larval neuromuscular performance is not altered significantly in slg A mutants. Metabolic pathways other than that involving proline oxidase are able to sustain glutamatergic synaptic function in Drosophila larvae.