We have defined a minimal Arabidopsis CATALASE 3 (CAT3) promoter sufficient to drive evening-specific circadian transcription of a LUCIFERASE reporter gene. Deletion analysis and site-directed mutagenesis reveal a circadian response element, the evening element (EE: AAAATATCT), that is necessary for evening-specific transcription. The EE differs only by a single base pair from the CIRCADIAN CLOCK ASSOCIATED 1-binding site (CBS: AAAAAATCT), which is important for morning-specific transcription. We tested the hypothesis that the EE and the CBS specify circadian phase by site-directed mutagenesis to convert the CAT3 EE into a CBS. Changing the CAT3 EE to a CBS changes the phase of peak transcription from the evening to the morning in continuous dark and in light-dark cycles, consistent with the specification of phase by the single base pair that distinguishes these elements. However, rhythmicity of the CBS-containing CAT3 promoter is dramatically compromised in continuous light. Thus, we conclude that additional information normally provided in the context of a morning-specific promoter is necessary for full circadian activity of the CBS.The circadian clock enables an organism to specifically partition aspects of its biology to precise times over the day (Dunlap, 1999). Although the circadian clock is, by definition, endogenous and continues to run in the absence of external time cues, environmental stimuli such as light and temperature act to entrain the internal processes of an organism both to the exact external daily period and in a defined relationship, or phase angle, to the diurnal cycle. For example, in Arabidopsis, light and temperature information are integrated to partition physiological activities such as circadian-regulated leaf movement, stomatal opening, and gene expression to distinct times of day or phases (McClung et al., 2002).A central theme that has emerged in circadian biology is that the core oscillator is composed of a negative feedback loop grounded in positive and negative transcriptional regulation (Dunlap, 1999). It has recently been demonstrated that the Arabidopsis circadian clock entails such a transcriptional feedback loop (Alabadí et al., 2001) that includes at least three components: TIMING OF CAB EXPRESSION 1 (TOC1; also called Arabidopsis PSEUDO-RESPONSE REGULATOR 1, APRR1; Millar et al., 1995;, CIRCADIAN CLOCK ASSOCIATED 1 (CCA1; Wang and Tobin, 1998), and LATE ELON-GATED HYPOCOTYL (LHY; Schaffer et al., 1998).CCA1 and LHY are single-Myb domain transcription factors, and DNA-binding activity of CCA1 to a CCA1-binding site (CBS: AAAAATCT) has been characterized (Wang et al., 1997). The hypothesized role of TOC1 as a transcription factor is based on similarity to CONSTANS, although DNA binding by TOC1 has not been experimentally established (Strayer et al., 2000). However, TOC1 (APRR1) has been shown to bind to PHYTOCHROME-INTERACTING FACTOR 3 (PIF3), a Myc-related basic helix-loop-helix transcription factor, and to the related PIF3-LIKE 1 (PIL1; Makino et al., 2002). Expression o...