Gymnotiform weakly electric fish produce electric organ discharges (EODs) that function in electrolocation and communication. The command signal for the EOD is produced by the medullary pacemaker nucleus, which contains two well-characterized neuron types: pacemaker cells and relay cells. In this study, we characterized a third neuron type in the pacemaker nucleus. These neurons, which we have named parvocells, were smaller (7-15 microm in diameter) than relay and pacemaker cells. The parvocells were labeled with an antibody against the neuronal calcium-binding protein, parvalbumin, and were not labeled with several glial-specific antibodies. Parvocells had one to three fine processes that often terminated at the periphery of relay and pacemaker cell bodies. The parvalbumin-positive terminals of the parvocells colocalized with immunoreactivity for SV-2, suggesting that the parvocells form chemical synapses on the relay and pacemaker cells. Parvalbumin-positive neurons are frequently gamma-aminobutyric acid (GABA)ergic or glycinergic, and the cytoplasm of the parvocell somata was immunoreactive with a glycine antibody. Antibodies against glycine receptors and gephyrin, however, did not label any cells in the pacemaker nucleus, suggesting that the pacemaker nucleus does not contain glycine or GABA((A)) receptors. Electron microscopy revealed gap junctions between the membranes of parvocells and adjacent terminal-like structures. Furthermore, neurobiotin injected into individual pacemaker or relay cells labeled parvocells as well as other pacemaker and relay cells, demonstrating that the parvocells are dye-coupled to the other neuron types in the pacemaker nucleus. These findings indicate that the parvocells are histochemically distinct from relay and pacemaker cells and that they receive electrotonic inputs from and make chemical synapses back onto pacemaker and relay cells. Further study is needed to investigate the function of these neurons in regulating the EOD.