Mutations that abolish expression of an X-linked gene, FMR1, result in the pathogenesis of fragile X syndrome, the most common form of inherited mental retardation. To understand the normal function of the FMR1 protein, we have produced fly strains bearing deletions in a Drosophila homolog of FMR1 (dfmr1). Since fragile X patients show a number of abnormal behaviors including sleep problems, we investigated whether a loss-of-function mutation of dfmr1 affect circadian behavior. Here we show that under constant darkness (DD), a lack of dfmr1 expression causes arrhythmic locomotor activity, but in light:dark cycles, their behavioral rhythms appear normal. In addition, the clock-controlled eclosion rhythm is normal in DFMR1-deficient flies. These results suggest that DFMR1 plays a critical role in the circadian output pathway regulating locomotor activity in Drosophila.