Casein kinase 1ε (CK1ε) plays an important regulatory role in various cellular processes including circadian rhythms. Mutations in CK1ε or the recognition site on its substrate PER2 result in modulation of the circadian period length. In particular, the tau mutation (R178C) in the catalytic domain of CK1ε was identified as the molecular basis for a dose-dependent heritable shortened circadian period in hamsters. However, the biochemical basis for the physiological effects of the tau mutant remains unclear. It has been reported that the tau mutation has reduced in vitro activity against some substrates but increased in vitro activity against other substrates. To better understand the effects of the CK1ε tau mutation, an ATP-phosphopeptide conjugate was synthesized to yield a transition-state bisubstrate analog. Kinase activity assays determined that the tau mutant has 80% reduced activity and a fourfold decrease in sensitivity to the bisubstrate analog compared to wild type. This confirms that Arg178 is important in the recognition of the preferred phosphosubstrates of CK1ε.