To study the neurochemical identity of axons in synaptic contact with identified hypothalamic neurosecretory neurons in rats, we combined retrograde axonal transport of a marker molecule with postembedding immunogold staining for amino acid neurotransmitters. After intravenous injections of horseradish peroxidase, neurosecretory neurons with axons in the median eminence or neurohypophysis transported the peroxidase retrogradely back to the cell body of origin. Serial ultrathin sections from the paraventricular and arcuate nuclei were immunostained with glutamate or GABA antisera. Peroxidase-labeled neurons and their dendrites received synaptic contact from colloidal gold-labeled axons immunoreactive for GABA or for glutamate. Axons which were highly immunoreactive for GABA and other axons immunoreactive for glutamate but not for GABA consistently made converging synaptic contact with the same peroxidase-labeled cell. Some of the peroxidase-labeled neurons from the arcuate nucleus which were postsynaptic to both GABA and glutamate axons were themselves identified as being GABA immunoreactive. Serial ultrathin sections revealed that multiple presynaptic axons immunoreactive for glutamate or GABA made repeated contacts with single neurons. These results suggest a widespread convergence of the major inhibitory and excitatory amino acid transmitter on the neurons which control both the anterior and posterior pituitary hormones.