Serotonin (5-HT) plays an important role in circadian rhythms, acting to modulate photic input to the mammalian clock, the suprachiasmatic nucleus (SCN), as well as playing a role in non-photic input. The transcription factor Pet-1 is an early developmental indicator of neurons that are destined for a 5-HTergic fate. Mice lacking the Pet-1 gene show a 70% loss of 5-HT immunopositive cell bodies in adult animals. 5-HT neurotoxic lesion studies using 5,7-dihydroxytryptamine (5,7-DHT) have highlighted species-specific differences in response to 5-HT depletion and studies using knockout mice lacking various 5-HT receptors have helped to elucidate the role of individual 5-HT receptors in mediating 5-HT's effects on circadian rhythms. Here we investigate the effects of a developmental disruption of the 5-HT system on the SCN and circadian wheel-running behavior. Immunohistochemical analysis confirmed depletion of 5-HT fiber innervation to the SCN as well as greatly reduced numbers of cell bodies in the raphe nuclei in Pet-1 knockout mice. These mice also display significantly longer free-running periods than wildtype or heterozygote counterparts. In light-dark cycles, knockouts showed a shift in peak wheel running behavior towards the late night as compared to wildtype and heterozygote animals. When kept in constant darkness for 70 days, wildtype animals showed decreases in free-running period over time while the period of knockout animals remained constant. Immunohistochemical analysis for neuropeptides within the SCN indicates that the behavioral changes observed in Pet-1 knockout mice were not due to gross changes in SCN structure. These results suggest that developmental loss of serotonergic input to the clock has long-term consequences for both circadian clock parameters and the temporal organization of activity.