Backround & Aims: We investigated the effect of the NHE3-binding PDZ-adapter proteins NHERF1, E3KARP and PDZK1 on NHE3 transport rate regulation in native murine colon. Methods: NHE3 transport rates were assessed fluorometrically in the surface cell region of BCECF-loaded mur-ine colonic crypts. Results: In NHERF1-deficient surface enterocytes, acid-activated NHE3 transport rate was significantly reduced to 65% of wt, yet significant inhibition of NHE3 transport activity by forskolin, 8-Br-cGMP and ionomycin was observed. In E3KARP-deficient colonic enterocytes, inhibition by ionomycin was selectively abolished. In PDZK1-deficient surface enterocytes, acid-activated NHE3 transport rate was strongly reduced to 35% of that in wt enterocytes, and no significant inhibition of transport rate was seen by any of the above second messengers. In NHERF1 and E3KARP-deficient intestine, NHE3 BBM abundance was unchanged, whereas it was significantly decreased in PDZK1-deficient intestine. Conclusions: The results demonstrate that the PDZ-Proteins NHERF1, E3KARP and PDZK1 are important regulators of NHE3 transport activity in native murine intestinal epithelium and appear to serve different, and partly signal-specific functions.