During the estrous cycle and beginning in estrus, the mammary gland undergoes pregnancy-like development that depends on transcriptional regulation by the estrogen and progesterone receptors (ER, PR) and Pax-2 as well as the action of the growth factors Wnt-4 and RANKL. In this report, we first describe the decay and delayed expression of ERalpha, PR, and Pax-2 proteins as well as depression of Wnt-4 and RANKL mRNA coincident with the strong estrogen surge in proestrus. In time-course studies using ovari-ectomized mice, a single estrogen injection replicated these delays and caused an 18 h delay in Wnt-4 expression. Molecular time-delay systems are at the core of cellular cycles, most notably the circadian clock, and depend on proteasome degradation of transcriptional regulators that exhibit dedicated timing functions. The cytoplasmic dynamics of these regulators govern delay duration through negative transcription/translation feedback loops. A proteasome inhibitor, PS-341, blocked estrogen-stimulated ERalpha, PR, and Pax-2 decay and proteasome chymotryptic activity, assayed using a fluorogenic substrate, was elevated in proestrus correlating with the depletion of the transcription factors. The 18-h delay in Wnt-4 induction corresponded to the turnover time of Pax-2 protein in the cytoplasm and was eliminated in Pax-2 knockout mammary tissue, demonstrating that Pax-2 has a unique timing function. The patterns of estrogen-triggered ERalpha, PR, and Pax-2 turnover were consistent with a negative transcriptional feedback. Retarding the expression of ERalpha, PR, and Pax-2 may optimize preparations for pregnancy by coordinating expression of critical receptors and transcription factors with rising estrogen and progesterone levels in estrus. The estrogen surge in proestrus has no defined mammotropic function. This study provides the first evidence that it is a synchronizing signal triggering proteasome-dependent turnover of mammary gland ERalpha, PR, and Pax-2. We hypothesize that the delays reflect a previously unrecognized timing system, which is present in all ovarian target tissues.