Recent studies have shown that casein kinase I (CKI) is an essential regulator of the mammalian circadian clock. However, the detailed mechanisms by which CKI regulates each component of the circadian negative-feedback loop have not been fully defined. We show here that mPer proteins, negative limbs of the autoregulatory loop, are specific substrates for CKI and CKI␦. The CKI phosphorylation of mPer1 and mPer3 proteins results in their rapid degradation, which is dependent on the ubiquitin-proteasome pathway. Moreover, CKI and CKI␦ are able to induce nuclear translocation of mPer3, which requires its nuclear localization signal. The mutation in potential phosphorylation sites on mPer3 decreased the extent of both nuclear translocation and degradation of mPer3 that are stimulated by CKI. CKI and CKI␦ affected the inhibitory effect of mPer proteins on the transcriptional activity of BMAL1-CLOCK, but the inhibitory effect of mCry proteins on the activity of BMAL1-CLOCK was unaffected. These results suggest that CKI and CKI␦ regulate the mammalian circadian autoregulatory loop by controlling both protein turnover and subcellular localization of mPer proteins.In most living organisms, behavioral and physiological processes display ϳ24-h rhythms that are controlled by circadian pacemakers (6,15,22,37). Although these rhythms persist in constant conditions, fluctuations of the natural environment entrain rhythms to precisely 24-h periods. The circadian clock is made up of three components: an input pathway adjusting the time, a central oscillator generating the circadian signal, and an output pathway manifesting itself in circadian physiology and behavior (10,12,23,25). In mammals, the master circadian pacemaker is located in the suprachiasmatic nucleus, which controls neural and humoral signals that either drive output rhythms or synchronize peripheral oscillators with the day-night cycle (3,38).Autoregulatory feedback loops of gene expression are believed to provide the rhythm-generating mechanisms. Since several clock genes are conserved in flies and mammals, the fundamental mechanism may be evolutionarily conserved (40). In mammals, the positive limb of the feedback loop is composed of CLOCK and BMAL1 (2,4,8,16), while the negative limb is composed of cryptochrome proteins (Cry1 and Cry2) and period proteins (Per1, Per2, and Per3) (1,5,27,29,31,32,33,35,41).In Drosophila melanogaster, doubletime (dbt) was identified as a kinase, which is thought to phosphorylate dPer (Drosophila Period) (17, 21). Missense mutations in dbt result in an altered circadian rhythm. Null alleles of dbt result in hypophosphorylation of dPer and arrhythmia. Dbt-mediated phosphorylation destabilizes dPer so that the level of dPer increases only when dTim (Drosophila Timeless) is increasing (17,21,24). In the Syrian hamster, the tau mutation is a spontaneous, semidominant mutation causing a short-period phenotype. Using a positional syntenic cloning strategy, the tau locus is revealed to encode casein kinase I ε (CKIε), a mammalian homolog o...