The renal medulla, considered critical for the regulation of salt and water balance and long-term blood pressure control, is enriched in anandamide and two of its major metabolizing enzymes, cyclooxygenase-2 (COX-2) and fatty acid amide hydrolase (FAAH). Infusion of anandamide (15, 30 and 60 nmol/min/kg) into the renal medulla of C57BL/6J mice stimulated diuresis and salt excretion in a COX-2- but not COX-1-dependent manner. To determine if endogenous endocannabinoids in the renal medulla can elicit similar effects, the effects of intramedullary isopropyl dodecyl fluorophosphate (IDFP), which inhibits the two major endocannabinoid hydrolases, were studied. IDFP treatment increased the urine formation rate and sodium excretion in a COX-2- but not COX-1-dependent manner. Neither anandamide or IDFP affected the glomerular filtration rate. Neither systemic (0.625 mg/kg/30 min, intravenous) or intramedullary (15 nmol/min/kg/30 min) IDFP pretreatment prior to intramedullary anandamide (15-30 nmol/min/kg) strictly blocked effects of anandamide, suggesting that hydrolysis of anandamide was not necessary for its diuretic effect. Intramedullary IDFP had no effect on renal blood flow but stimulated renal medullary blood flow. The effects of IDFP on urine flow rate and medullary blood flow were FAAH-dependent as demonstrated using FAAH knockout mice. Analysis of mouse urinary prostaglandin E2 (PGE2) concentrations by high performance liquid chromatography-electrospray ionization tandem mass spectrometry showed that IDFP treatment decreased urinary PGE2. These data are consistent with a role of FAAH and endogenous anandamide acting through a COX-2-dependent metabolite to regulate diuresis and salt excretion in the mouse kidney.