Protein phosphorylation plays essential roles in eukaryotic circadian clocks. Like PERIOD in animals, the Neurospora core circadian protein FRQ is progressively phosphorylated and becomes extensively phosphorylated before its degradation. In this study, by using purified FRQ protein from Neurospora, we identified 43 in vivo FRQ phosphorylation sites by mass spectrometry analysis. In addition, we show that CK-1a and CKII are responsible for most FRQ phosphorylation events and identify an additional 33 phosphorylation sites by in vitro kinase assays. Whole-cell metabolic isotope labeling and quantitative MS analyses suggest that circadian oscillation of the FRQ phosphorylation profile is primarily due to progressive phosphorylation at the majority of these newly discovered phosphorylation sites. Furthermore, systematic mutations of the identified FRQ phosphorylation sites led to either long or short period phenotypes. These changes in circadian period are attributed to increases or decreases in FRQ stability, respectively. Together, this comprehensive study of FRQ phosphorylation reveals that regulation of FRQ stability by multiple independent phosphorylation events is a major factor that determines the period length of the clock. A model is proposed to explain how FRQ stability is regulated by multiple phosphorylation events. mass spectrometry ͉ casein kinase ͉ frequency E ukaryotic circadian oscillators from fungi to mammals are controlled by autoregulatory negative feedback loops (1-4). In the filamentous fungus Neurospora crassa, 2 protein complexes function in the core circadian negative feedback loop (5, 6). WHITE COLLAR complex (WCC), formed by WC-1 and WC-2, activates transcription of the frequency ( frq) gene by binding to its promoter (7-13). On the other hand, FFC (consisting of FRQ and the FRQ-interacting RNA helicase, FRH) inhibits WCC activity by promoting the phosphorylation, and consequently repression, of frq transcription (12,(14)(15)(16)(17)(18).Like the animal PERIOD (PER) proteins, FRQ is progressively phosphorylated after its synthesis and becomes extensively phosphorylated before its disappearance, resulting in a robust oscillation of its phosphorylation profile (19). One role of FRQ phosphorylation is to promote FRQ degradation through the ubiquitin-proteasome pathway mediated by ubiquitin E3 ligase SCF FWD-1 . FWD-1 acts as the substrate-recruiting subunit that recognizes and binds phosphorylated FRQ (20)(21)(22). Under normal conditions, FRQ is phosphorylated by CKII, and PKA (12,16,19,[23][24][25]. In the ck-1a (casein kinase 1a), cka (catalytic subunit of CKII), and ckb-1 (regulatory subunit of CKII) mutants, FRQ is hypophosphorylated and more stable relative to the wild type, resulting in arrhythmia or long-period phenotypes (12,23,25). These results suggest that CK-1a and CKII phosphorylate and promote FRQ degradation. In contrast, PKA counters the role of casein kinases by stabilizing FRQ (12,16). FRQ is also dephosphorylated and stabilized by protein phosphatases PP1 and PP4 (17,2...