The Period (PER), Timeless (TIM), and Double-Time (DBT) proteins are essential components of one feedback loop in theDrosophilacircadian molecular clock. PER and TIM physically interact. Coexpression of PER and TIM promotes their nuclear accumulation and influences the activity of DBT: although DBT phosphorylates and destabilizes PER, this is suppressed by TIM. Experiments usingDrosophilacells in culture have indicated that PER can translocate to the nucleus without TIM and will repress transcription in a DBT-potentiated manner. In this study, we examined the control of PER subcellular localization inDrosophilaclock cellsin vivo.We found that PER can translocate to the nucleus intim01null mutants but only if DBT kinase activity is inhibited. We also found that nuclear PER is a potent transcriptional repressor indbtmutantsin vivowithout TIM. Thus,in vivo, DBT regulates PER subcellular localization, in addition to its previously documented role as a mediator of PER stability. However, DBT does not seem essential for transcriptional repression by PER. It was reported previously that overexpression of a second kinase, Shaggy (SGG)/Glycogen Synthase Kinase 3, accelerates PER nuclear accumulation. Here, we show that these effects of SGG on PER nuclear accumulation require TIM. We propose a revised clock model that incorporates this tight kinase regulation of PER and TIM nuclear entry.