The hypothalamus is the crucial part of the brain that regulates homeostasis throughout the body. It governs the endocrine and autonomic nervous systems, temperature, heart rate, emotional and motivational states, reproduction, energy and water balances, and circadian rhythms. In contrast to the prevailing belief that hypothalamic neurons use peptides, neuromodulators, or other slow-acting agents as their principal neuroactive substances, we present data indicating that the primary excitatory transmitter released by medial hypothalamic neurons is glutamate. This surprising new evidence is based on three converging approaches: Immunogold cytochemistry revealed that some hypothalamic neurons and their processes in vitro contained high amounts of immunoreactive glutamate. Ca2+ digital video imaging showed that cytoplasmic Ca2+ levels of cultured neurons, elevated because of spontaneous presynaptic release of a hypothalamic transmitter, were reduced by perfusion with the selective glutamate receptor antagonists cyano-2,3-dihydroxy-7-nitroquinoxaline and 2-aminophosphonovaleric acid. Electrophysiological analysis of whole-cell patch-clamp recordings from single and pairs of monosynaptically coupled hypothalamic neurons in culture showed that virtually all spontaneous and evoked EPSPs appear to be mediated by synaptic secretion of glutamate.