In vitro experiments demonstrated the stimulating effect of the serum- and glucocorticoid-inducible kinase SGK1 on the activity of the Na+/H+ exchanger NHE3. Phosphoinositide dependent kinase PDK1 activates SGK1 and may similarly play a role in the regulation of NHE3-dependent epithelial electrolyte transport. To explore the role of PDK1 in the regulation of NHE3 activity hypomorphic mice expressing 10–25% of PDK1 (pdk1hm) were compared to their wild type littermates (pdk1wt). NHE3 activity in intestine and PDK1 overexpressing HEK293 cells has been estimated utilizing BCECF fluorescence. NHE activity was reflected by the Na+-dependent pH recovery from an ammonium pre-pulse ([Delta]pHnhe). DpHnhe was in pdk1hm mice only 30 ± 6 % of the value obtained in pdk1wt mice. Conversely, DpHnhe was 32 ± 7% larger in PDK1 overexpressing HEK cells than in cells expressing the empty vector. The NHE3 inhibitor S3226 (10 mM) completely abolished both the difference between pdk1hm and pdk1wt mice and between PDK1 overexpressing and empty vector transfected HEK cells, respectively. In conclusion, the results point to a role of PDK1-dependent signaling in the regulation of NHE dependent electrolyte transport.