The suprachiasmatic nucleus (SCN) is the central circadian pacemaker governing the circadian rhythm of locomotor activity in mammals. The mammalian retina also contains circadian oscillators, but their roles are unknown. To test whether the retina influences circadian rhythms of locomotor behavior, the authors compared the activity of bilaterally enucleated hamsters with the activity of intact controls held in constant darkness (DD). Enucleated hamsters showed a broader range of free-running periods (tau) than did intact hamsters held for the same length of time in DD. This effect was independent of the age at enucleation (on postnatal days 1, 7, or 28). The average tau of intact animals kept in DD from days 7 or 28 was significantly longer than that of intact animals kept in DD from day 1 or any of the enucleated groups. This indicates that early exposure to light-dark cycles lengthens the tau and that the eye is required to maintain this effect even in DD. These data suggest that hypothalamic circadian pacemakers may interact continuously with the retina to determine the tau of locomotor activity. Enucleation caused a large decrease in glial fibrillary acidic protein in the SCN but has no (or slight) effects on calbindin, neuropeptide Y, vasopressin, or vasoactive intestinal polypeptide, which suggests that enucleation does not produce major damage to the SCN, an interpretation that is supported by the fact that enucleated animals retain robust circadian rhythmicity. The presence of an intact retina appears to contribute to system-level circadian organization in mammals perhaps as a consequence of interaction between its circadian oscillators and those in the SCN.