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The circadian clock is driven by cell-autonomous transcription͞ translation feedback loops. The BMAL1 transcription factor is an indispensable component of the positive arm of this molecular oscillator in mammals. Here, we present a molecular genetic screening assay for mutant circadian clock proteins that is based on real-time circadian rhythm monitoring in cultured fibroblasts. By using this assay, we identified a domain in the extreme C terminus of BMAL1 that plays an essential role in the rhythmic control of E-box-mediated circadian transcription. Remarkably, the last 43 aa of BMAL1 are required for transcriptional activation, as well as for association with the circadian transcriptional repressor CRYPTO-CHROME 1 (CRY1), depending on the coexistence of CLOCK protein. C-terminally truncated BMAL1 mutant proteins still associate with mPER2 (another protein of the negative feedback loop), suggesting that an additional repression mechanism may converge on the N terminus. Taken together, these results suggest that the C-terminal region of BMAL1 is involved in determining the balance between circadian transcriptional activation and suppression.circadian clock ͉ real-time monitor T he mammalian circadian clock is a highly dynamic system that generates periodic fluctuations in the mRNA expression levels of hundreds of genes to confer near 24-h rhythmicity to behavior, physiology, and metabolic processes, thereby allowing mammals to anticipate the momentum of the day (1). The master clock resides in the suprachiasmatic nuclei (SCN) of the brain and, in turn, synchronizes circadian clocks in peripheral tissues (2). Even fibroblasts in culture contain an active circadian clock that has the same genetic makeup of the central clock in the SCN (3-6). To keep pace with the day-night cycle, the SCN clock, but not peripheral clocks, are entrained by light.Circadian rhythms are generated by a molecular oscillator that consists of intertwined positive and negative transcription͞ translation feedback loops involving a set of clock genes (7) and clock-controlled output genes that link the oscillator to clockcontrolled processes (8). BMAL1 (MOP3) and CLOCK are basic helix-loop-helix PAS transcription factors that heterodimerize and (by means of binding to E-box promoter elements) transactivate the Period (Per1 and Per2) and Cryptochrome (Cry1 and Cry2) genes and an orphan nuclear receptor Rev-Erb␣ core oscillator gene. Subsequently, PER and CRY proteins act as negative elements by inhibiting the activity of the CLOCK͞BMAL1 heterodimer, whereas REV-ERB␣ negatively regulates Bmal1 gene expression (1, 9). The above feedback mechanism is supported by biochemical, molecular, and genetic evidence; however, formal proof of its requirement in the maintenance of circadian clock oscillations has not been shown thus far.Genetic ablation of mBmal1 results in complete disruption of the mammalian circadian clock at the behavioral and molecular levels (10). However, except for the PAS elements, which are required for association with CLOCK (11)...