Dec2, a member of the basic helix-loop-helix superfamily, is a recently confirmed regulatory protein for the clockwork system. Transcripts of Dec2, as well as those of its related gene Dec1, exhibit a striking circadian oscillation in the suprachiasmatic nucleus, and Dec2 inhibits transcription from the Per1 promoter induced by Clock/Bmal1 [Honma, Kawamoto, Takagi, Fujimoto, Sato, Noshiro, Kato and Honma (2002) Nature (London) 419, 841-844]. It is known that mammalian circadian rhythms are controlled by molecular clockwork systems based on negative-feedback loop(s), but the molecular mechanisms for the circadian regulation of Dec2 gene expression have not been clarified. We show here that transcription of the Dec2 gene is regulated by several clock molecules and a negative-feedback loop. Luciferase and gel retardation assays showed that expression of Dec2 was negatively regulated by binding of Dec2 or Dec1 to two CACGTG E-boxes in the Dec2 promoter. Forced expression of Clock/Bmal1 and Clock/Bmal2 markedly increased Dec2 mRNA levels, and up-regulated the transcription of the Dec2 gene through the CACGTG E-boxes. Like Dec, Cry and Per also suppressed Clock/Bmal-induced transcription from the Dec2 promoter. Moreover, the circadian expression of Dec2 transcripts was abolished in the kidney of Clock/Clock mutant mice. These findings suggest that the Clock/Bmal heterodimer enhances Dec2 transcription via the CACGTG E-boxes, whereas the induced transcription is suppressed by Dec2, which therefore must contribute to its own rhythmic expression. In addition, Cry and Per may also modulate Dec2 transcription.