We show that the cell cycle-regulated transcription of the TATA-less cdc25C gene in late S/G2 is largely mediated by a novel promoter element (CDE) located directly 5' to one of the two major transcription initiation sites. Genomic dimethylsulfate footprinting experiments, using either synchronized or sorted normally cycling cells, show the formation in vivo of a CDE-protein complex in both G0 and G1 cells and its dissociation in G2. Mutation of the CDE severely impairs cell cycle regulation of the cdc25C promoter and results in high expression in G0/G1, indicating that the CDE functions as a cell cycle-regulated cis-acting repressor element. Cell cycle regulation is also lost upon removal of the enhancer region located immediately upstream of the CDE, but is largely restored when this enhancerless minimal cdc25C promoter fragment is linked to the constitutive SV40 early enhancer. This indicates that the CDE is dependent on the presence of a transcriptional enhancer to effect cell cycle regulation. Our observations suggest that the periodic activation of the cdc25C gene in late S/G2 is brought about, at least in part, by a unique regulatory mechanism involving the cell cycle-regulated dissociation of a repressor from the CDE.