1. Intracellular recordings from magnocellular neurons in the supraoptic nucleus (SON) were obtained from rat hypothalamic slices to determine the effects of specific transmitter antagonists on evoked postsynaptic potentials (PSPs), action potential after-discharge, and spontaneously occurring PSPs. 2. Broad-spectrum excitatory amino acid (EAA) antagonists, kynurenic acid (KYN) and gamma-d-glutamylglycine (DGG), significantly diminished or eliminated electrically evoked depolarizing PSPs and spike discharges. These compounds also greatly reduced the amplitude and frequency of spontaneous PSPs. 3. The specific N-methyl-D-aspartate (NMDA) receptor antagonist, DL-2-amino-5-phosphonopentanoic acid (AP5), did not significantly reduce these measures of synaptic activation under these experimental conditions. 4. The gamma-aminobutyric acid (GABA) antagonist, bicuculline methiodide (BIC), partially antagonized some PSPs when the cells were hyperpolarized (-75 to -80 mV) with steady injected currents; KYN antagonized BIC-resistant PSPs. 5. The involvement of a hypothetical cholinergic input to the SON in the responses to stimulation of the region dorsolateral to the SON was tested by bath application of nicotinic cholinergic antagonists, particularly d-tubocurarine (dTC). Nicotinic cholinergic antagonists, even after prolonged exposure to high concentrations, did not block the responses of SON cells to dorsolateral stimulation. 6. These findings strongly suggest that EAAs mediate fast excitatory synaptic responses of SON neurons to stimulation of cells and axons in the region dorsolateral to the SON. The blockade of almost all spontaneous EPSPs by broad-spectrum EAA antagonists likewise argues that EAAs are responsible for the majority of ongoing fast excitatory input. These responses appear to involve an interaction with kainate- and/or quisqualate-type EAA receptors.