Glutamate (Glu) and γ-aminobutyric acid (GABA) are the most abundant excitatory and inhibitory neurotransmitters in the mammalian hypothalamus. Glu and GABA-containing neurons have both been shown to synapse with gonadotropin-releasing hormone (GnRH) and other neuroendocrine systems in the hypothalamus of several species. Since their direct interactions could play a pivotal role in governing neuroendocrine function, we performed double-label immunostaining for Glu and for glutamic acid decarboxylase (GAD) as a marker for GABAergic neurons in hypothalamic sections from adult female cynomolgus monkeys. Ultrastructural analysis of 785 Glu-immunoreactive (-ir) and GAD-ir elements in the medial septum (MS), arcuate nucleus-ventral hypothalamic tract (VHT1), supraoptic nucleus (SON), paraventricular nucleus (PVN), and median eminence (ME) revealed that 63% were Glu-ir, 28% were GAD-ir, and 9% were Glu+GAD-ir. In addition, we observed surprisingly consistent labeling of 2-4% somata (SOM), 65-80% dendrites (DEN), and 15-30% axons and terminals (AXO) in all of these areas. Characterization of 177 interactions (36% synapses, 64% contacts) by pre-/post-transmitter content indicated that 29% contained Glu/GAD, 15% Glu/Glu, and 15% Glu/Glu+GAD, while 16% were unlabeled/Glu, 9% were unlabeled/GAD, and 16% expressed other transmitter combinations. Regional analysis of these interactions showed that 43% occurred in the MS, 22% in VHT1, 14% in SON, 9% in PVN, and 12% in the ME. AXO/DEN interactions made up 51 % of all labeled interactions characterized, and were comprised 29% of Glu/GAD, 22% of Glu/Glu, and 18% of the Glu/Glu+GAD type. AXO/DEN synapses were more prevalent than contacts in all areas except the PVN and of course the ME, where anatomical synapses do not occur. AXO/SOM interactions represented approximately 15% of all those identified, and were predominantly unlabeled/Glu (71 %) and unlabeled/GAD (18%) synapses. Almost all (95%) AXO/SOM synapses and 75% of the contacts occurred in the MS. DEN/DEN interactions, 28% of the total, were composed 50% of Glu/GAD, 12% of Glu/Glu, and 18% of the Glu/Glu+GAD type. The relatively few DEN/ DEN synapses all appeared in the MS, whereas much more abundant DEN/DEN contacts were more widely distributed. DEN/SOM interactions, 6% of the total, appeared only as contacts, with the majority (60%) again located in the MS. In addition, the MS contained 48% of all asymmetrical synapses (vs. 35% in VHT1 and 17% in SON), 62% of all symmetrical synapses (vs. 19%inVHTl and 14% in SON), and 35%of all contacts (vs. 21% in VHT1 and 12% in SON) identified. In summary, Glu-ir neuronal processes are about twice as numerous as GAD-ir elements in the female monkey hypothalamus. Glu-ir afferents are found in nearly 2/3 of these contacts, participating in classical synapses, novel specializations, and intimate local circuits. Interactions occur principally in the MS, VHT1, and to a lesser extent in the SON, PVN, and ME, all of which are important sites of neuroendocrine integration. On the basis of our past and present data, we propose a model of Glu-GABA-GnRH neurointeractions which may help mediate GnRH secretion in the monkey hypothalamus.