In vitro experiments demonstrated the stimulating effect of the serum- and glucocorticoid-inducible kinase SGK1 on the activity of the Na⁺/H⁺ exchanger NHE3. SGK1 requires activation by phosphoinositide dependent kinase PDK1 which may thus similarly play a role in the regulation of NHE3-dependent epithelial electrolyte transport. The present study has been performed to explore the role of PDK1 in the regulation of NHE3 activity. As mice completely lacking functional PDK1 are not viable, hypomorphic mice expressing approx. 20 % of PDK1 (pdk1^hm) were compared to their wild type littermates (pdk1^wt). NHE3 activity in intestine and PDK1 overexpressing HEK293 cells has been estimated utilizing BCECF fluorescence of intracellular pH. NHE activity was reflected by the Na⁺-dependent pH recovery from an ammonium pre-pulse (pHnhe). The pH changes following an ammonium pulse allowed the calculation of cellular buffer capacity which was not significantly different between pdk1^hm and pdk1^wt mice. pHnhe was in pdk1^hm mice only 30 ±6 % of the value obtained in pdk1^wt mice. Conversely, pHnhe was 32 ±7% larger in PDK1 overexpressing HEK cells than in HEK cells expressing the empty vector. The difference between pdk1hm and pdk1^wt mice and between PDK1 overexpressing and empty vector transfected HEK cells, respectively, was completely abolished in the presence of NHE3 inhibitor S3226 (10 µM). In conclusion, defective PDK1 expression leads to significant impairment of NHE3 activity in intestine pointing to a role of PDK1-dependent signaling in the regulation of NHE mediated electrolyte transport.