The secretion of Cl- by the epithelial cells of the distal colonic crypt provides the driving force for the movement of fluid into the luminal space of the colon. Experimental procedures were performed on colonic epithelium harvested from Sprague-Dawley rats following humane sacrifice by cervical dislocation. The distal colon was removed post mortem and cut into sections. The epithelium was then dissected away from the underlying muscle layer or else crypts were detached from the tissue by Ca2+ depletion and then isolated. 17β-estradiol (E2) stimulates a rapid and sustained suppression of this secretory process in the female but not in male rat colon. The modulation of KCNQ1 channel activity through E2-induced PKCδ isozyme and PKA isoform I activation regulates the electrochemical gradient that facilitates apical Cl- secretion. The sexual dimorphism in the E2 response is associated with elevated expression of PKCδ in female compared to the male colon tissue. The anti-secretory response to E2 is regulated throughout the rat female reproductive (oestrous) cycle and is primed by genomic regulation of the kinases. E2 (1-10 nM) decreased cAMP-dependent secretion in colonic epithelia during the oestrus, meta-oestrus and dioestrus stages of the cycle. Weaker inhibition of secretion was demonstrated in the pro-oestrus stage. The expression levels of PKCδ and PKA fluctuated throughout the oestrous cycle and correlated with the potency of E2 in suppressing secretion. E2 stimulated the up-regulation of PKCδ and PKA expression through increased transcription via a PKCδ-MAPK-CREB regulated pathway indicating a genomic priming of the anti-secretory response. PKCδ was activated by the membrane impermeant E2-BSA and this response was inhibited by the oestrogen receptor (ER) antagonist ICI 182,780. The 66kDa ERα isoform was present at the plasma membrane of female colonic crypt cells with a lower abundance found in male colonic crypts. This study demonstrates acute and chronic E2 regulation of intestinal secretion through interdependent transcriptional and signalling responses.