In mammals, circadian rhythms in physiology and behaviour are regulated by a master clock located in the suprachiasmatic nucleus (SCN) of the hypothalamus. The SCN controls the rhythmic release of melatonin from the pineal gland and this is one mechanism through which the SCN can communicate clock information to the brain and body. Melatonin exerts its effects via two classes of G-protein coupled receptors which are expressed in a number of brain areas including the SCN, hippocampus, dorsomedial hypothalamus, and habenula. Melatonin is known to inhibit SCN neuronal activity, but surprisingly, the neurophysiological actions of melatonin in other brain areas remain largely unexplored. One important brain region to study is the habenula, since it conveys the forebrain inputs to the mid-brain structures and has also been proposed as a component of the brain’s circadian system. Here, we used whole-cell patch clamp electrophysiology to determine the actions of melatonin on neurons in the medial habenula (MHb) of the mouse. Coronal brain slices (~250 μm thick) containing the habenula were prepared during the day from young adult mice (4-10 weeks of age) that were housed under a 12h light:12h dark (LD) cycle. Slices were maintained in a submersion chamber mounted on a microscope and superfused with oxygenated artificial cerebrospinal fluid (aCSF). MHb neurons were visualized and targeted for conventional whole-cell current clamp recordings. Baseline activity was assessed and the effects of addition of melatonin to the aCSF determined. Under control conditions, MHb neurons (n=245) showed a range of resting membrane potential (RMP), from highly depolarised (-23mV) to moderately hyperpolarised states (-46mV), with a mean RMP of -35±0.4 mV. Highly depolarised cells tended to be silent, not producing action potentials, while spontaneously discharging neurons displayed a range of action potential firing rate (0.2 – 31.4 Hz). There was no day-night difference in the RMP or action potential production frequency. Application of melatonin (1 nM) elicited responses from ~ 80% of all MHb neurons tested (n=70). Responses ranged from hyperpolarizations in RMP and suppression of action potential production to depolarizations and elevations in firing rate. Collectively, these data reveal that the majority of MHb neurons are responsive to exogenous melatonin, raising the possibility that melatonin can act through the MHb to modulate brain and physiological functions.