A major mechanism of electroneutral NaCl absorption in the human ileum and colon involves coupling of Na(+)/H(+) and Cl(-)/HCO(3)(-) exchangers. Disturbances in these mechanisms have been implicated in diarrheal conditions. Probiotics such as Lactobacillus have been indicated to be beneficial in the management of gastrointestinal disorders, including diarrhea. However, the molecular mechanisms underlying antidiarrheal effects of probiotics have not been fully understood. We have previously demonstrated Lactobacillus acidophilus (LA) to stimulate Cl(-)/HCO3- exchange activity via an increase in the surface levels and expression of the Cl(-)/HCO3- exchanger DRA in vitro and in vivo. However, the effects of LA on NHE3, the Na(+)/H(+) exchanger involved in the coupled electroneutral NaCl absorption, are not known. Current studies were, therefore, undertaken to investigate the effects of LA on the function and expression of NHE3 and to determine the mechanisms involved. Treatment of Caco2 cells with LA or its conditioned culture supernatant (CS) for 8-24 h resulted in a significant increase in Na(+)/H(+) exchange activity, mRNA, and protein levels of NHE3. LA-CS upregulation of NHE3 function and expression was also observed in SK-CO15 cells, a human colonic adenocarcinoma cell line. Additionally, LA treatment increased NHE3 promoter activity, suggesting involvement of transcriptional mechanisms. In vivo, mice gavaged with live LA showed significant increase in NHE3 mRNA and protein expression in the ileum and colonic regions. In conclusion, LA-induced increase in NHE3 expression may contribute to the upregulation of intestinal electrolyte absorption and might underlie the potential antidiarrheal effects of probiotics.