The secretion of Cl- in the distal colonic cells across the apical membrane provides the driving force for the movement of fluid into the luminal space. In the case of cAMP dependent secretion, basolateral KCNQ1 channels carry out the K+ recycling required to establish a favourable membrane electrical potential for the secretion of Cl-. We have previously documented that 17β-estradiol (E2) inhibited Cl- secretion via targeting of KCNQ1 channels via the activation of the novel PKCδ isozyme and PKA isoform I (1) The inhibition of secretion was observed in female mucosa only and was due to a higher expression level of PKCδ compared to the male tissue. The present study revealed the anti-secretory response to bacterial toxins is regulated throughout the rat reproductive (estrous) cycle and is primed by genomic regulation of the protein kinases. The study was carried out in distal colonic epithelia from Sprague-Dawley rats. Short circuit current (Isc) provided a measure of Cl- secretion. E2 (1-10 nM) decreased Isc stimulated by bacterial toxins in colonic epithelia during the estrus, metaestrus and diestrus stages of the estrous cycle. A weak inhibition was demonstrated in the proestrus stage and no inhibition of secretion was observed in the male tissue. The basal expression levels of PKCδ and PKA fluctuated throughout the estrous cycle. Increased expression of both kinases correlated with an increased anti-secretory effect of E2. Further studies demonstrated that PKCδ and PKA were up-regulated at a transcriptional level via a PKCδ-MAPK-CREB regulated pathway indicating a priming of the anti-secretory response by estrogen. PKCδ was activated by the membrane impermeant E2-BSA and this response was inhibited by the ER antagonists tamoxifen and ICI 182,780. The 66KDa ERα isoform was shown to be present at the plasma membrane of female colonic crypts and was absent from male crypts. This study demonstrates estrogen regulation of fluid secretion in the rat distal colon both at a rapid acute level and a transcriptional level, demonstrating an interdependent relationship between both non-genomic and genomic hormone signaling mechanisms.