The steroid hormone, 17β-Oestradiol (E2), has been shown to modulate electrolyte balance in the distal colon via a non-genomic mechanism (McNamara et al, 1995). We have previously reported the rapid E2 regulation of Na⁺/H⁺ exchange and K⁺ channels, which requires the upstream activation of complex kinase pathways including Protein Kinase C (PKC) (Clondiffe et al, 2001) and Mitogen Activated Protein Kinases (MAPK). Recently we reported the activation of Protein Kinase D (PKD) a known downstream target of PKC (O’Mahony et al, 2003). This study sought to identify the signalling molecules stimulated rapidly in response to E2 (10nM) and resulting protein-protein kinase interactions. Sprague-Dawley rats (3 months old) were sacrificed by cervical dislocation and distal colonic crypts were isolated. The oestrous cycle stage was determined by cervical smear. PKC phosphorylation was determined by western blotting using specific phospho-antibodies. Protein complexes were detected by co-immunoprecipitation. PKD autophosphorylation was detected by an in vitro kinase assay (IVKA). Protein Kinase A (PKA) activation was investigated using a non-radioactive PepTag Assay (Promega). We report for the first time a gender specific activation of PKCα (2 fold, 5min) and PKCε (2 fold, 3min) in female distal colonic crypts with no activation noted in male rat colonic crypts. We demonstrated PKA activation (4 fold, 5min) in the female with no activation noted in male tissue. IVKA analysis of E2-induced autophosphorylation of PKD showed an increase in activity at the oestrous stage (1 fold, 15min) with a decrease at the dioestrous stage (2 fold, 5min). This differential PKD regulation may be due to the oestrogen background of the female rat. Co-immunoprecipitation of PKD complexes showed an association with PKCδ (5 min) and P38 MAPK (15 min) with no association noted in the male tissue. PKCδ also associated with P38 MAPK (2 fold, 15min) specific to female tissue indicating a complex between PKCδ/PKD/P38 MAPK. PKD did not associate with PKCε or PKA in the female or male distal colonic crypt. In conclusion, we provide the first evidence for the differential activation of PKC and the oestrous cycle dependent activation of PKD in the female distal colonic crypt. This study demonstrated the formation of multi-protein complexes in response to E2 specific to the female rat colon. The downstream ion channels targets of these complexes remains to be elucidated.