Neurons of the lateral hypothalamus that contain the peptide transmitters orexins/hypocretins are essential for sustaining normal consciousness, and their loss leads to narcolepsy. The firing of orexin neurons varies according to states of consciousness and energy balance (1). Orexin cells are hyperpolarised in response to physiological increases of glucose. However, their responses to other macronutrients have not been studied, although the protein:carbohydrate ratio of a meal affects arousal (2). We investigated effects of dietary amino acids on orexin cells using patch-clamp recordings in transgenic mouse brain slices, where orexin neurons are identified by targeted expression of green fluorescent protein. Values are means ± S.E.M., compared by student’s t-test. We found that a mixture of amino acids (AAs), based on previously published in vivo microdialysis data from rat hypothalamus (3), depolarised orexin cells by 12.6 ± 0.7 mV (n = 25). This effect is opposite to that of glucose and, furthermore, the presence of AAs suppressed the glucose-induced hyperpolarisation (physiologically relevant transition from 1 to 5 mM glucose caused 18.2 ± 1.0 mV hyperpolarisation in control conditions vs 7.6 ± 1.0 mV in presence of AA mix, n = 5 for each condition, P < 0.001). The current voltage (I-V) relationship of the AA-induced depolarising conductance change revealed that a current with a reversal potential (Erev) of -97.7 ± 2.9 mV (n = 8) was inactivated, suggesting that it was caused at least in part by inactivation of a K+ conductance (in our conditions Erev,K = -107.6 mV). Our evidence indicates that the AA induced depolarisation is mediated in part by activation of Na+ coupled, electrogenic “system A” AA transporters and by increase of intracellular ATP and subsequent closure of ATP-sensitive K+ channels. This was indicated by partial inhibition of the response by ATP-sensitive K+ channel inhibitor tolbutamide (6.7 +- 1.2 mV depolarisation, n = 9, P < 0.001). The I-V relationship of the net inhibited current in presence of tolbutamide had no reversal potential, suggesting that the remaining current is mediated by an electrogenic transporter. The response was completely abolished by combined application of system A transporter blocker, N-(methylamino)isobutyric acid, and tolbutamide (-0.2 ± 0.5 mV change in membrane potential, n = 9, P < 0.001). These data indicate that orexin neurons are capable of generating a signal that non-linearly integrates availability of energy (glucose) and structural (amino acids) substrates. Since the brain concentrations of both glucose (4) and AAs (3) change after feeding, this might be a way for the brain to sense how balanced a meal is.