The structural and functional heterogeneity of the kidney and fast transient changes in signaling molecules present a tremendous experimental challenge to study variety of functional processes in whole organ. Extracellular nucleotides such as adenosine, 5′-triphosphate (ATP) are essential local signaling molecules in many organs including the kidney. Goal of this study was to develop quantitative approach for detection of hydrogen peroxide (H2O2) and ATP in freshly isolated whole organs. We established and described a novel approach to measure acute concentrations changes in endogenous substances in the freshly isolated perfused kidney. This method is based on using enzymatic microelectrode biosensors and utilizes standard amperometry technique. In addition to measurements of basal levels of H2O2 and ATP, this technique allows rapid detection of purinergic signaling, and immediately provides observation of changes and fluctuations in ATP concentration in freshly isolated organs during vehicle or drug perfusion. The real time changes in ATP and H2O2 concentrations in response to angiotensin II (Ang II), a pluripotent peptide hormone of the renin-angiotensin system, were measured. Freshly isolated kidneys of the Sprague Dawley (SD) or Dahl salt-sensitive (SS) rats were flushed with 1 µM Ang II under constant laminar perfusion. For these experiments rats were anesthetized with isoflurane inhalation and the surgical procedure was performed on a temperature controlled table. The kidneys were perfused through the distal aorta with a HBSS to clear blood from the organs. The flushing was continued for 2-3 min until the kidneys were completely blanched, then the animal was euthanized by pneumothorax. The left kidney was then removed and aorta ligated above the renal artery for the consequent perfusion. After surgery, the flushed kidney with catheterized renal vessels was placed into the bath solution and swathed to the silicone resin to avoid movement during perfusion. Dual simultaneous amperometric recordings were made with the biosensors detecting ATP and H2O2 levels. Biosensors were obtained from Sarissa Biomedical Ltd (Coventry, UK). Ang II perfusion produced a rapid simultaneous release of both ATP and H2O2 in the kidney cortex. Substance release rate and total integral release were also estimated. Controls with AT1 receptor antagonist losartan and catalase revealed specificity of used biosensors. Renal cortex interstitial H2O2 concentration was also determined in SS rats fed low or high salt diets by traditional microdialysis and analyzed with Amplex red to compare these two approaches. The data indicate that SS rats have increased levels of H2O2 compared to SD rats, especially when fed a high salt diet. Furthermore, Ang II induced ATP and H2O2 release, which can stimulate arteriolar vasoconstriction or effect renal tubular transport in the kidney, and this effect is also enhanced in SS rats. In conclusion, current method is a unique powerful approach for substance release detection during pathological or normal conditions in whole organs including the kidney.