The study of the functional processes in the whole kidney presents a tremendous experimental challenge due to the structural and functional heterogeneity of the kidney tissues and rapid transient changes in the concentrations of the signaling molecules. The purines, like adenosine-5’-triphosphate (ATP) as well as reactive oxygen species including H2O2 are among the most important paracrine signaling agents. Angiotensin II (AngII) directly regulates many processes in the body and is strongly linked to the hypertension. We recently developed a novel approach based on the use of enzymatic biosensors suitable for real-time measurements of endogenous substances release in freshly isolated rat kidney. AngII-induced changes of interstitial concentration of ATP and H2O2 were determined in Sprague Dawley (SD) and Dahl salt sensitive (SS) rats. Freshly isolated kidneys of the SD or SS rats were infused with AngII (1 μM) under constant laminar flow. The maximum amplitude of the AngII induced H2O2 responses averaged 70 ± 15, 189 ± 58 and 301 ± 91 nM for SD and SS rats fed a low (0.4%) and high salt (4%; 3 weeks) diets, respectively. The H2O2 response exhibited a slow increase to a stable maximal level that lasted during the rest of the perfusion time. The average production rate was 67 ± 17, 187 ± 55 and 294 ± 81 nM/s for SD, and SS rats fed low and high salt diets. The kinetics of ATP release had different characteristics compared to H2O2 release with rapid increase in concentration triggered by AngII and followed by a slow decay of the signal due to ATP conversion to adenosine. The maximum amplitudes averaged 449 ± 145, 630 ± 49 and 1087 ± 96 nM for SD rats and SS rats fed low and high salts. The ATP production rates were 257 ± 100, 496 ± 202 and 829 ± 319 nM/s for the corresponding groups. To test AT receptor subtype mediated the acute release of ATP and H2O2 in response to AngII, we used an AT1 receptor antagonist losartan. Losartan (10 μM) inhibited the release of both ATP and H2O2 in response to AngII application. Thus, these experiments reveal that established approach can be successfully used to analyze acute changes in endogenous substances in response to drug perfusion. Importantly, these data demonstrate that perfusion of the kidney with AngII rapidly induced release of both ATP and H2O2.