Endogenous near 24h (circadian) rhythms in physiology and behaviour are generated by the main circadian clock in the hypothalamic suprachiasmatic nucleus (SCN). The synchronization of the SCN clock by environmental light (photic cues) and by stimuli that promote internal arousal (non-photic cues) results in daily rhythms in sleep and wake, core body temperature, etc. Some components of the molecular basis for the SCN clock (the so-called clock genes/proteins) are well characterized and include the period 1 (per1) gene and its protein product PER1. The major light input pathway to the SCN utilizes glutamate and the effects of glutamate on mouse SCN neurons, including those expressing an enhanced destabilized green fluorescent protein (EGFP) driven by the per1 promoter are known. The neurochemicals communicating non-photic information to the SCN are less well understood, but there has been considerable interest in the arousal-promoting orexin/hypocretin neuropeptides. Orexin is synthesized by discrete populations of neurons localized mainly in the lateral hypothalamus. Some lateral hypothalamic neurons innervate the SCN region but it is unclear if and how orexins influence SCN neuronal activity, particularly those expressing per1. We used whole-cell recordings to investigate the effects of orexin on SCN neurons expressing per1::EGFP as well as those neurons in which per1::EGFP could not be detected (‘per1’, and ‘non-per1’ neurons, respectively) in SCN brain slices in vitro. In the majority (70%) of per1 and non-per1 neurons tested, orexin (50-300 nM) induced biphasic responses, causing the membrane potential of the neurons to oscillate. In some of these neurons, co-application of orexin with gabazine (20 µM) suppressed a phasic GABA-mediated hyperpolarization to reveal a subtle orexin-induced depolarization. In 5% of neurons examined, orexin caused membrane depolarization, while in 10% robust hyperpolarization was seen. This orexin-induced hyperpolarization was inhibited by pretreatment with gabazine or tetrodotoxin (500 nM), suggesting that orexin acts presysnaptically to recruit GABAergic interneurons. Voltage-clamp recordings supported this, and revealed a dose-dependent increase in inhibitory postsynaptic current frequency during bath application of orexin that was eliminated by gabazine. We also observed that orexin caused a significantly stronger inhibition (p< x 10-5) in night per1 and non-per1 neurons when compared with day neurons. This night-time orexin-induced hyperpolarization in per1 and non-per1 neurons was robust (-70 mV) and long lasting(≈45 min). We conclude that orexin has complex effects on SCN neurons that involve the recruitment of local GABAergic interneurons that tonically or phasically inhibit the neurons. These effects had a clear day/night difference.