The Drosophila period protein (PER) is a predominantly nuclear protein and a likely component of a circadian clock. PER is required for daily oscillations in the transcription of its own gene and thus participates in a circadian feedback loop. In this study, key pacemaker neurons of the Drosophila brain were examined to determine whether the subcellular distribution of PER changes with the time of day. Indeed, PER was found to accumulate in the cytoplasm for several hours before entering the nucleus during a narrow time window. Three long-period mutations (perL) cause a delay in the timing of nuclear translocation and a further delay at elevated temperature. The data indicate that regulation of PER nuclear entry is critical for circadian oscillations by providing a necessary temporal delay between PER synthesis and its effect on transcription.