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We assessed the mechanisms by which specialized hypothalamic ventromedial nucleus (VMN) neurons utilize both glucose and long-chain fatty acids as signaling molecules to alter their activity as a potential means of regulating energy homeostasis. Fura-2 calcium (Ca²⁺) and membrane potential dye imaging, together with pharmacological agents, were used to assess the mechanisms by which oleic acid (OA) alters the activity of dissociated VMN neurons from 3- to 4-wk-old rats. OA excited up to 43% and inhibited up to 29% of all VMN neurons independently of glucose concentrations. In those neurons excited by both 2.5 mM glucose and OA, OA had a concentration-dependent effective excitatory concentration (EC₅₀) of 13.1 nM. Neurons inhibited by both 2.5 mM glucose and OA had an effective inhibitory concentration (IC₅₀) of 93 nM. At 0.5 mM glucose, OA had markedly different effects on these same …