The tau mutation is a semidominant autosomal allele that dramatically shortens period Length of circadian rhythms in Syrian hamsters. We report the molecular identification of the tau Locus using genetically directed representational difference analysis to define a region of conserved synteny in hamsters with both the mouse and human genomes. The tau Locus is encoded by casein kinase I epsilon (CKl epsilon), a homolog of the Drosophila circadian gene double-time. in vitro expression and functional studies of wild-type and tau mutant CKl epsilon enzyme reveal that the mutant enzyme has a markedly reduced maximal velocity and autophosphorylation state. In addition, in vitro CKl epsilon can interact with mammalian PERIOD proteins, and the mutant enzyme is deficient in its ability to phosphorylate PERIOD. We conclude that tau is an allele of hamster CKl epsilon and propose a mechanism by which the mutation Leads to the observed aberrant circadian phenotype in mutant animals.