The maize (Zea mays L.) Opaque2 (O2) protein is an endosperm-specific transcriptional activator whose DNA-binding activity is regulated diurnally by a phosphorylation/dephosphorylation mechanism. We show that the O2 transcript undergoes pronounced oscillations during the day-night cycle. The highest level of the O2 message is present at midday and the lowest level at midnight. The level of O2 transcript follows a diurnal rhythm that appears controlled by the circadian clock. Two different endosperm-expressed DNA-binding proteins, PBF (prolamin box-binding factor) and OHP1 (O2-heterodimerizing protein 1), were also analyzed. While the PBF message levels oscillate diurnally, the steady-state levels of OHP1 transcript were constant through the day and night. We present data showing that the seed is not directly involved in the perception of the light signal, but presumably responds to diurnal fluxes of nutrients into the endosperm. Moreover, we show that the O2 protein is not involved in the regulation of its own transcript levels. These data indicate that O2 activity is down-regulated at night by both a reduction in O2 transcript and by hyperphosphorylation of residual O2 protein, and suggest that regulatory gene activity during endosperm development may be acutely sensitive to a diurnal signal(s) emanating from the plant and passing into the developing seeds.