Very little is known about the circadian regulation of cell entry into the S and M phases of the cell cycle. Yet, in the mouse esophagus, a seven- to ninefold increase in DNA synthesis coincides with nocturnal feeding. The phosphorylation of the cAMP response element binding protein (CREB), a transcriptional factor, may regulate hypothalamic circadian rhythms in the brain. Here, we investigate the circadian regulation of CREB and Ser-133-phospho-CREB (PCREB) in the mouse esophagus by immunocytochemical and biochemical methods. We found that, during the dark phase, coincident with the onset of feeding and increased DNA synthesis, esophageal CREB and PCREB expression decreased. Although CREB-like immunoreactivity (CREB-lir) was expressed in many different cell types, it was concentrated in the mucosa, particularly in the replicating basal cell layer. The injection of epidermal growth factor, at a dosage known to maximally stimulate esophageal DNA synthesis in a 4- to 8-h period, rapidly decreased PCREB levels within 10 min of injection. We speculate that PCREB-lir may be involved in the circadian regulation of cell cycle events in the intact mouse esophagus.