The neurotransmitters mediating the synaptic interactions among the neurons of the pyloric system of the stomatogastric ganglion (STG) of the lobster, Panulirus interruptus, were examined using a combination of electrophysiological, pharmacological, and biochemical techniques. Iontophoretically applied L-glutamate inhibited all motor neurons of the pyloric system. This inhibitory response was blocked by low concentrations of picrotoxin but unaffected by atropine. The anterior burster (AB) interneuron, pyloric dilator (PD) motor neurons, and ventricular dilator (VD) motor neuron were depolarized and excited by iontophoretically applied acetylcholine (ACh). The lateral pyloric (LP) and pyloric (PY) constrictor motor neurons were inhibited by ACh and by the cholinergic agonist, carbachol. These inhibitory cholinergic responses were blocked by atropine but not by picrotoxin. The inhibitory postsynaptic potentials (IPSPs) evoked by the constrictor motor neurons were blocked by picrotoxin but not by atropine. Taken together with previously published data (15, 18), this suggests that the constrictor motor neurons release glutamate at both their excitatory neuromuscular junctions and their inhibitory intraganglionic junctions. The lucifer yellow photoinactivation technique (27) was used to study separately the neurotransmitters released by the electrically coupled PD and AB neurons. The AB-evoked IPSPs were blocked by picrotoxin but not by atropine. The PD-evoked IPSPs were blocked by atropine and other muscarinic antagonists but not by picrotoxin. Somata of PD neurons contained choline acetyltransferase (CAT) activity, but somata of AB neurons contained no detectable CAT activity. On the basis of the data in this paper and previously published data (17, 18), we conclude that the PD neurons release ACh at both their excitatory neuromuscular junctions and their inhibitory intraganglionic connections. Although the AB neuron is electrically coupled to the PD neurons, the AB neuron is not cholinergic. Glutamate is a likely transmitter candidate for the AB neuron. These data show that electrically coupled neurons can release different transmitters. Furthermore, these data show that an IPSP can be the result of the combined actions of two different neurotransmitters, each released from a different neuron. The functional consequences of these conclusions are explored in the following papers (9, 22).