Probiotics are viable nonpathogenic microorganisms that are considered to confer health benefits to the host. Recent studies indicated that some Lactobacillus species function as probiotics and have been used as alternative treatments for diarrhea, which occurs due to increased secretion, decreased absorption, or both. However, the direct effects of probiotics on intestinal electrolyte absorption are not known. Therefore, we examined the effects of Lactobacillus on luminal chloride/hydroxyl (Cl(-)/OH(-)) exchange activity in human intestinal epithelial cells. Postconfluent Caco-2 cells were treated with the Lactobacillus species Lactobacillus acidophilus (LA), Lactobacillus casei, Lactobacillus plantarum, or Lactobacillus rhamnosus (LR) for 3 h at a multiplicity of infection of 50. Cl(-)/OH(-) exchange activity was measured as 4,4'-diisothiocyanostilbene-2, 2'-disulfonic acid-sensitive (36)Cl uptake in base-loaded cells. Treatment with live, but not heat-killed, LA and LR significantly increased Cl(-)/OH(-) exchange activity (approximately 50%), whereas other species were ineffective. Similarly, the conditioned medium (supernatant) of live LA increased Cl(-)/OH(-) exchange. The ability of LA or its conditioned culture medium to enhance Cl(-)/OH(-) exchange activity was blocked by PI-3 kinase inhibition but was unaffected by inhibition of mitogen-activated protein kinases. Corresponding to the increased Cl(-)/OH(-) exchange activity, LA treatment increased the surface expression of the apical anion exchanger, SLC26A3 [Down Regulated in Adenoma (DRA)]. The increased DRA membrane localization might contribute to the increased Cl(-) absorption by LA. Our results suggest that LA secretes soluble effector molecule(s) into the culture medium that stimulate apical Cl(-)/OH(-) exchange activity via phosphatidylinositol-3 kinase mediated mechanism.