A striking feature of the circadian clock is its flexible yet robust response to various environmental conditions. To analyze the biochemical processes underlying this flexible-yet-robust characteristic, we examined the effects of 1,260 pharmacologically active compounds in mouse and human clock cell lines. Compounds that markedly (>10 s.d.) lengthened the period in both cell lines, also lengthened it in central clock tissues and peripheral clock cells. Most compounds inhibited casein kinase I epsilon (CKI epsilon) or CKI epsilon phosphorylation of the PER2 protein. Manipulation of CKI epsilon/delta dependent phosphorylation by these compounds lengthened the period of the mammalian clock from circadian ( 24 h) to circabidian ( 48 h), revealing its high sensitivity to chemical perturbation. The degradation rate of PER2, which is regulated by CKI epsilon/delta-dependent phosphorylation, was temperature-insensitive in living clock cells, yet sensitive to chemical perturbations. This temperature-insensitivity was preserved in the CKI epsilon/delta-dependent phosphorylation of a synthetic peptide in vitro. Thus, CKI epsilon/delta-dependent phosphorylation is likely a temperature-insensitive period-determining process in the mammalian circadian clock.