Although much is known about the regulation of the circadian rest–activity cycle by the hypothalamic suprachiasmatic nucleus in nocturnal rodents, little is known about the neural substrates that regulate the temporal organization of nocturnal activity within the active phase. In this report, data are presented in Syrian hamsters to implicate the habenula – believed to be involved in motivation, reward and motor control – as a candidate site for such a role. First, by examining hamsters during the day and night and by introducing a ‘novel’ running wheel in order to induce daytime motor activity, we showed that immunoreactive c-Fos expression in the lateral and medial habenula is related to motor activity/arousal. Second, by transecting the habenula’s major efferent pathway (fasciculus retroflexus), we showed that the interruption of habenula neural output alters the daily amount of motor activity, lengthens the period of the circadian rest–activity rhythm and disrupts the species-typical pattern of nocturnal motor activity, measured as either wheel-running behavior or general locomotor activity. Instead of the usual pattern of night-time locomotion, characterized by a prolonged bout of elevated activity in the early night followed by shorter sporadic bouts or the cessation of activity altogether, lesioned animals exhibited a more homogeneous, undifferentiated temporal profile extending across the night. These data suggest a previously unrecognized function of the habenula whereby it regulates the temporal pattern of activity occurring within a circadian rest–activity window set by the suprachiasmatic nucleus.