Adult and larval insects are rapidly anesthetized by carbon dioxide (CO2); however, the mechanisms have not been addressed. In this study, we use larval Drosophila to investigate the actions of CO2 to explain the behavioral effects of rapid immobilization and cardiac arrest with acute exposure to CO2. To determine if the central nervous system (CNS) is required, studies were performed with and without the CNS. The effects of low pH induced by exposure to CO2 were also examined. An acidic saline increases the heart rate in contrast to saline containing CO2. Synaptic transmission at the skeletal neuromuscular junction (NMJ) is blocked by CO2 but not by low pH. The site of action is postsynaptic by a decreased sensitivity to glutamate, the neurotransmitter at Drosophila NMJs. The CNS remains active in synaptic transmission when exposed to CO2 which is in contrast to the synapses at the NMJ. In summary, the effects of CO2 are directly mediated on the heart to stop it and at skeletal NMJs by a reduced sensitivity to glutamate, the released neurotransmitter, from the motor nerve terminals. The rapid behavioral and physiological effects cannot be accounted for by action on the CNS within the larvae nor by a pH effect indirectly induced by CO2. The glutamate receptors in the D. melanogaster preparation are similar in function to ionotropic glutamate receptors in vertebrates which could account for the observational phenomena of CO2 not yet explained mechanistically in vertebrates.