The circadian clock enables the anticipation of daily recurring environmental changes by presetting an organism's physiology and behavior. Driven and synchronized by a central pacemaker in the brain, circadian output genes fine-tune a wide variety of physiological parameters in peripheral organs. However, only a subset of circadianly transcribed genes seems to be directly regulated by core clock proteins. Assuming that yet unidentified transcription factors may exist in the circadian transcriptional network, we set out to develop a novel technique, differential display of DNA-binding proteins (DDDP), which we used to screen mouse liver nuclear extracts. In addition to several established circadian transcription factors, we found DNA binding of heat-shock factor 1 (HSF1) to be highly rhythmic. HSF1 drives the expression of heat-shock proteins at the onset of the dark phase, when the animals start to be behaviorally active. Furthermore, Hsf1-deficient mice have a longer free-running period than wild-type littermates, suggesting a combined role for HSF1 in the mammalian timekeeping and cytoprotection systems. Our results also suggest that the new screening method DDDP is not limited to the identification of circadian transcription factors but can be applied to discover novel transcriptional regulators in various biological systems.