Background: Trafficking, brush border membrane (BBM) retention, and signal-specific regulation of the Na+/H+ exchanger 3 (NHE3) is regulated by the Na+/H+ Exchanger Regulatory Factor (NHERF) family of PDZ-adapter proteins, which enables the formation of multiprotein complexes. We previously reported a differential association of the NHERFs to the lipid raft and non-raft fraction of NHE3 in murine intestinal BBM. Aim: This study was undertaken to explore the association of NHE3 and NHERF2 with the cGMP-dependent kinase II (cGKII), a key enzyme in hormonal and toxin-mediated inhibition of NHE3, within lipid rafts, and to assess the effect of stimulating this signal transduction pathway on the raft assembly of NHE3, NHERF2 and cGKII in vivo. Methods: Murine BBM was isolated from wildtype, NHE3-deficient, cGKII-deficient, and NHERF2-deficient mice before and after intestinal application of a heat-stable Escherichia coli toxin (STa)/guanylin analogue, which binds to and activates guanylate cycle C (GCC) in vivo, followed by cervical dislocation 2 hours after drug application and removal of the small intestine. Lipid raft and non-raft fractions were separated by Optiprep density gradient centrifugation of Triton X solubilised isolated small intestinal BBM. The raft-associated and non-raft proteins were precipitated and studied by Western analysis. Results: NHE3 and NHERF2 were strongly lipid raft-associated. The cGMP-dependent kinase II, which together with NHERF2 is essential for STa/guanylin-mediated NHE3 inhibition, was found in two molecular entities, a larger band of ~85 KD, which was exclusively lipid raft-associated, and a smaller band of ~72 KD that was mostly non-raft associated. NHERF2 deletion resulted in a decreased lipid raft association of NHE3, but not of cGKII. The application of an oral STa/guanylin analogue to the mice and subsequent small intestinal BBM isolation and lipid raft flotation assay demonstrated a redistribution of cGKII, with an increase of the lipid raft associated larger size cGKII, more cosegregation of NHE3, NHERF2 and cGKII in the same raft fraction, and a decrease of the smaller size, non-raft cGKII. Conclusion: The differential association of the NHERF2, as well as cGKII, with the raft-associated and the non-raft fraction of NHE3 in the brush border membrane is likely one component of STa/guanylin-mediated inhibition of NHE3. Many players of the signalling pathway for the STa/guanylin analogues via cGKII, leading to NHE3 inhibition, are associated with lipid rafts in the murine small intestine, and stimulation of this signalling pathway causes more redistribution of the cGKII to the lipid rafts, possibly by lipid modification.