Epoxyeicosatrienoic acids (EETs) are arachidonic acid metabolites formed endogenously via the cytochrome P450 pathway in rat, rabbit, and human kidney. We characterized the effects of the four regioisomeric EETs on ion transport in the renal epithelial cell line, LLC-PK1. Among the EETs, 14,15-EET was the most potent inhibitor of 86Rb uptake. Its effect was concentration-dependent (IC50 = 75 nM) and stereoselective to the 14S, 15R-EET. Experiments measuring 14,15-EET-induced 86Rb uptake inhibition in the presence of inhibitors of Na(+)-K(+)-ATPase activity (ouabain), Na(+)-K(+)-Cl- cotransporter (furosemide), and Na(+)-H+ exchanger (amiloride) suggested that 14,15-EET inhibits ion transport via an amiloride-sensitive mechanism. These results, together with previous reports demonstrating their endogenous production in the kidney, suggest an important role for EETs, specifically 14,15-EET, in the regulation of ion and water reabsorption in the kidney and implicate their function in renal pathophysiology.