Heavy water (D2O) lengthens the period of free-running circadian rhythms in most organisms. We compared the effect of D2O on free-running locomotor activity rhythms in intact and SCN-lesioned (SCN-X) hamsters that had recovered circadian rhythmicity following implantation of SCN grafts. The animals were housed individually in cages equipped with running wheels, and locomotor activity was monitored using a computer-based data acquisition system. At the end of the behavioral tests, animals were anesthetized and perfused. Brain sections were immunostained for vasoactive intestinal peptide (VIP) and vasopressin (VP) to evaluate the extent of the lesion and the presence of a functional graft. The D2O similarly lengthened the period of free-running activity without affecting amount of activity in both intact and in SCN-X grafted animals. The results indicate that D2O acts directly on the SCN to lengthen the free-running period, and suggest that coupling between pacemakers within the grafted SCN is as efficient as in the intact SCN.