A wide range of processes in plants, including expression of certain genes, is regulated by endogenous circadian rhythms. The circadian clock-associated 1 (CCA1) and the late elongated hypocotyl (LHY) proteins have been shown to be closely associated with clock function in Arabidopsis thaliana. The protein kinase CK2 can interact with and phosphorylate CCA1, but its role in the regulation of the circadian clock remains unknown. Here we show that plants overexpressing CKB3, a regulatory subunit of CK2, display increased CK2 activity and shorter periods of rhythmic expression of CCA1 and LHY. CK2 is also able to interact with and phosphorylate LHY in vitro. Additionally, overexpression of CKB3 shortened the periods of four known circadian clock-controlled genes with different phase angles, demonstrating that many clock outputs are affected. This overexpression also reduced phytochrome induction of an Lhcb gene. Finally, we found that the photoperiodic flowering response, which is influenced by circadian rhythms, was diminished in the transgenic lines, and that the plants flowered earlier on both long-day and short-day photoperiods. These data demonstrate that CK2 is involved in regulation of the circadian clock in Arabidopsis. C ircadian rhythms are driven by endogenous biological clocks that regulate many biochemical, physiological, and behavioral processes in a wide variety of organisms. According to current models, circadian clocks regulating these rhythms consist of input pathways, a central oscillator, and output pathways (1-3). Oscillators are thought to generate rhythms by a transcription-translation negative feedback loop (4-8). Studies in cyanobacteria, Neurospora, Drosophila, and mouse have found that both positive and negative elements that activate and inhibit the transcription of clock genes are required to maintain the loop. In addition, posttranscriptional and posttranslational regulation play important roles in circadian clocks in Drosophila and Neurospora (7-10). The oscillator can be entrained by input pathways from environmental cues such as light and temperature, and, in turn, regulates specific cellular events such as expression of clock-controlled genes (1-3).Until recently, little was known about the molecular mechanisms of circadian clocks in plants (11-13). In Arabidopsis thaliana, the toc1 mutation affects the period of many circadian rhythms (14,15). Although the corresponding gene has not yet been cloned, it is thought that TOC1 encodes a component of the oscillator. The ELF3 gene is proposed to act in the input pathway (16). Two Myb-related genes, circadian clock-associated 1 (CCA1) and late elongated hypocotyl (LHY), have also been identified as potential clock genes (17, 18), and CCA1 was found to act as a transcription factor for Lhcb gene expression (19). Expression of CCA1 and LHY oscillates with a circadian rhythm. Constitutive expression of CCA1 was shown to abolish several distinct circadian rhythms, suppress its own expression as well as that of LHY, and delay flowering substa...