Antisera specific for gamma-aminobutyric acid (GABA) or its biosynthetic enzyme, glutamate decarboxylase, were used in pre- and postembedding immunocytochemical techniques at the light and electron microscopic levels, to visualize the GABAergic innervation of the hypothalamic supraoptic nucleus. Immunostaining for glutamate decarboxylase or gamma-aminobutyric acid were also combined with oxytocin and vasopressin immunolocalization, thereby permitting evaluation of the contribution of the innervation onto each type of neuron in this nucleus. Light microscopy of semithin plastic sections or vibratome slices stained for glutamate decarboxylase or gamma-aminobutyric acid, with peroxidase-antiperoxidase as immunolabel, revealed an extensive punctate labeling in the supraoptic nucleus and its immediate surroundings. Quantitative analysis of glutamate decarboxylase immunostaining in semithin sections indicated a comparable density of immunopositive punctae at the anterior and posterior levels of the nucleus (14-27 X 10(6) per mm3 tissue). Glutamate decarboxylase- or gamma-aminobutyric acid-immunoreactive cell bodies were never observed within the nucleus although they were detected in the hypothalamus immediately dorsolateral to the nucleus. Electron microscopy of vibratome slices treated with antiglutamate decarboxylase or antigamma-aminobutyric acid and peroxidase-antiperoxidase, or of ultrathin sections stained directly with antigamma-aminobutyric acid and immunoglobulin-coupled colloidal gold, showed that the immuno-reactive punctae represented, in the main, axonal terminals. They invariably contained small, rounded clear vesicles and, at times, one or two larger, dense cored vesicles; they all formed symmetrical synapses onto magnocellular cell bodies and dendrites. Oxytocin and vasopressin neurons were contacted in a similar fashion by glutamate decarboxylase- or gamma-aminobutyric acid-positive boutons in semithin sections of the nucleus stained simultaneously for glutamate decarboxylase and oxytocin and in ultrathin sections stained for glutamate decarboxylase or gamma-aminobutyric acid and oxytocin or vasopressin. Glutamate decarboxylase- or gamma-aminobutyric acid-positive terminals often formed synapses onto two postsynaptic elements in the same plane of section ("double" synapses), a synaptic configuration usually encountered in supraoptic nuclei of lactating animals. In such cases, the postsynaptic somata were oxytocinergic.(ABSTRACT TRUNCATED AT 400 WORDS)