We assessed the mechanisms by which specialized hypothalamic ventromedial nucleus (VMN) neurons utilize both glucose and long chain fatty acids as signalling molecules to alter their activity as a potential means of regulating energy homeostasis. Fura-2 calcium (Ca²⁺) and membrane potential dye imaging, together with pharmacologic agents, were used to assess the mechanisms by which oleic acid (OA) alters the activity of dissociated VMN neurons from 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 55.3 nM. At 0.5 mM glucose, OA had markedly different effects on these same neurons. Inhibition of carnitine palmitoyl transferase, reactive oxygen species formation, long chain acetyl CoA synthetase and ATP-sensitive K⁺ channel activity or activation of UCP2 accounted for only ~20% of OA's excitatory and ~40% of its inhibitory effects. Inhibition of CD36, a fatty acid transporter that can alter cell function independently of intracellular fatty acid metabolism, reduced the effects of OA by up to 45%. Thus, OA affects VMN neuronal activity through multiple pathways. In glucosensing neurons, its effects are glucose-dependent. This glucose-OA interaction provides a potential mechanism whereby such "metabolic sensing" neurons can respond to differences in the metabolic states associated with fasting and feeding.