The different PDZ-adaptors of the NHERF family differentially associate with intestinal electrolyte transporters and modulate their activity in an agonist-specific fashion. STa is one of the most common causative agents in travellers diarrhea as well as diarrheal disease in livestock. It induces secretory diarrhea via binding to the guanylate cyclase C, eliciting cGMP production, resulting both in a stimulation of CFTR-mediated anion secretion and an inhibition of NHE3-mediated fluid absorption, and these effects are modulated by different NHERF adaptor proteins in cell lines. We investigated whether and how well the in vitro mediation of STa action on NHE3, and CFTR by different NHERF proteins and by the cyclic GMP kinase II (cGKII) in murine intestine correlates with their in vivo involvement in modulation of fluid transport. 10-7M of the STa analogue linacolide was applied to the luminal bath of chambered isolated jejunal mucosa of CFTR-, NHE3-, cGKII-, and NHERF1-3 deficient and WT mice, and short circuit current (Isc) response was measured. The same concentration of linaclotide was added to the jejunal perfusate during single pass perfusion of a jejunal segment in isoflurane-anesthetized, acid/base- and blood pressure controlled KO and WT mice, and net fluid balance before and after linaclotide application assessed gravimetrically. The peak Isc response to linaclotide was absent in CFTR KO, reduced by 88% in cGKII KO, by 40% in NHERF1 KO, and not significantly in NHERF2 and NHERF3 KO isolated mucosa. The magnitude of change from jejunal fluid absorption to secretion was reduced by 51% in cGKII KO, 49% in CFTR KO, 45% in NHERF 1 KO, 34% in NHERF2 KO, and not significantly in NHERF3 KO anesthetized mice. NHE3 deletion (48%) or pharmacological NHE3 inhibition (50%) reduced the magnitude of change to a similar degree as CFTR deletion. The results demonstrate that Isc measurements in isolated small intestinal mucosa are excellent models to selectively study agonist-mediated CFTR activation, but do not accurately predict in vivo secretory responses. Apart from direct interference with guanylate cyclase C activation, potential pharmacological strategies to prevent STa-mediated fluid loss may be targeting both NHERF1 and NHERF2.