The relationship between the endocannabinoid system in the renal medulla and the long-term regulation of blood pressure is not well understood. To investigate the possible role of the endocannabinoid system in renomedullary interstitial cells, mouse medullary interstitial cells (MMICs) were obtained, cultured and characterized for their responses to treatment with a selective inhibitor of fatty acid amide hydrolase (FAAH), PF-3845. Treatment of MMICs with PF-3845 increased cytoplasmic lipid granules detected by Sudan Black B staining and multilamellar bodies identified by transmission electron microscopy. HPLC analyses of lipid extracts of MMIC culture medium revealed a 205nm-absorbing peak that showed responsiveness to PF-3845 treatment. The biologic activities of the PF-3845-induced product (PIP) isolated by HPLC were investigated in anesthetized, normotensive surgically-instrumented mice. Intramedullary and intravenous infusion of PIP at low dose rates (0.5-1 AU/10 min) stimulated diuresis and natriuresis, whereas at higher doses, these parameters returned toward baseline but mean arterial pressure (MAP) was lowered. Whereas intravenous bolus doses of PIP stimulated diuresis, GFR and medullary blood flow (MBF) and reduced or had no effect on MAP, an intraperitoneal bolus injection of PIP reduced MAP, increased MBF, and had no effect on urinary parameters. Genetic or pharmacological ablation of the cannabinoid type 1 receptors in mice completely abolished the diuretic and vasodepressor properties of intramedullary infused PIP, suggesting that the PF-3845-induced product requires the presence of CB1 receptors in order to elicit its renal effects. In a radioactive competition binding assay, using Chinese hamster ovary cells expressing CB1 receptors, PIP successfully displaced the CB1 selective inverse agonist [3H] SR141716A, revealing that the lipid extract was able to compete for binding to CB1 receptors. Finally, we investigated the tubular location of diuretic activity that the PF-3845-induced lipid fraction exhibits. In a renal function in vivo experiment, we pre-treated anesthetized mice with an intramedullary infusion of one of four well-known diuretics. This procedure was followed by an intramedullary infusion of PIP (1AU). Only inhibition of the proximal tubule sodium reabsorption diminished the diuretic activity of the PF-3845-induced product, suggesting that the lipid fraction requires a physiologically intact proximal tubular reabsorption mechanism for it to produce diuresis. These data support a model whereby PF-3845 treatment of MMICs results in increased secretion of a neutral lipid which acts directly to promote diuresis and natriuresis and indirectly through metabolites to produce vasodepression. Efforts to identify the structure of the PF-3845-induced lipid and its relationship to the previously proposed renomedullary antihypertensive lipids are ongoing.