Medial hypothalamic neurons in pentobarbital anesthetized rats were identified by location and response to electrical stimulation of the amygdala, medial preoptic area, midbrain periaqueductal gray and median eminence. Cells were then examined for their sensitivity to microiontophoretic applications of GABA, glycine and 3 related amino acids, i.e. beta-guanidinopropionic acid, delta-aminovaleric acid and beta-alanine. Application of all agents decreased the spontaneous and glutamate or aspartate evoked activity of the majority of neurons in all identified categories. The majority of neurons were more sensitive to beta-guanidinopropionate, delta aminovalerate and GABA than to glycine and beta-alanine. Bicuculline and picrotoxin produced a selective and reversible antagonism of depressions evoked by GABA and GABA-like amino acids whereas strychnine produced a selective and reversible antagonism of depressions evoked by glycine and beta-alanine. Bicuculline and picrotoxin, but not strychnine, were observed to diminish synaptic inhibition evoked by electrical stimulation of several sites when these agents were administered by microiontophoresis and by i.v. injections in convulsive doses. These observations suggest that many medial hypothalamic neurons have both GABA and glycine receptors but that GABA may have the more important role as a neurotransmitter common to afferent or recurrent inhibitory hypothalamic pathways.