BACKGROUND & AIMS: Diarrhea results from reduced net fluid and salt absorption caused by an imbalance in intestinal absorption and secretion. The bulk of sodium and water absorption in the intestine is mediated by Na+/H+ exchanger 3 (NHE3), located in the luminal membrane of enterocytes. We investigated the effect of lysophosphatidic acid (LPA) on Na+/H+ exchanger activity and Na+-dependent fluid absorption in the intestine. METHODS: We analyzed the effects of LPA on fluid absorption in intestines of wild-type mice and mice deficient in Na+/H+ exchanger regulatory factor 2 (NHERF2; Nherf2(-/-)) or LPA 2 (Lpa(2)(-/-)). Roles of LPA5 and NHERF2 were determined by analysis of heterologous expression. RESULTS: Under basal conditions, LPA increased fluid absorption in an NHE3-dependent manner and restored the net fluid loss in a mouse model of acute diarrhea. Expression of the LPA receptor LPA5 was necessary for LPA-induced stimulation of NHE3 activity in colonic epithelial cells. Stimulation of NHE3 by the LPA-LPA5 signaling required coexpression of NHERF2, which interacted with LPA5. LPA-mediated intestinal fluid absorption was impaired in Nherf2(-/-) mice, demonstrating the requirement for NHERF2 in LPA5 activity. However, fluid absorption was unaltered in Lpa(2)(-/-) mice. LPA stimulated NHE3 and fluid absorption in part by increasing NHE3 protein abundance at the brush border membrane of intestinal epithelial cells. CONCLUSIONS: LPA is a potent stimulant of NHE3 and fluid absorption in the intestine, signaling through LPA5. Regulation by LPA5 depends on its interaction with NHERF2. LPA might be useful in the treatment of certain diarrheal diseases.