We aim to develop a cultured cell model, to serve as a system with which the altered circadian phenotypes produced by the clock gene variations could be studied in vitro. Tau mutation, which shortens the circadian period of hamsters and mice, was introduced into the CK1epsilon locus of cultured Rat1-R12 cells by gene targeting mediated by a recombinant adeno-associated virus (rAAV) vector. After transduction of Rat1-R12 cells with rAAV, about 0.14% of the drug-resistant cells underwent gene targeting at CK1epsilon locus. Of the three clones isolated, only one carried the targeted allele of tau mutation and two carried the targeted wild-type allele. The clone with the targeted tau mutant allele exhibited a significantly shorter circadian period compared to the clone with targeted wild-type allele. rAAV-mediated gene targeting in cultured somatic cells is a convenient and powerful tool for analyzing the phenotypic outcome of clock gene variations, and for elucidating the pathogenesis of the disorders associated with abnormal circadian rhythmicity.