Calcium flux in the podocytes is critical for normal and pathophysiological regulation of this type of cells, and excessive calcium signaling results in podocytes damage and improper glomeruli function. Purinergic activation of P2 receptors is a powerful and rapid signaling process; however, the exact physiological identity of P2 receptors subtypes in podocytes remains essentially unknown. The goal of this study was to determine P2 receptors profile in podocytes of the intact Sprague Dawley rat glomeruli using available pharmacological tools. Glomeruli were isolated by differential sieving, loaded with Fluo-4/Fura Red cell permeable calcium indicators and purinergic response in the podocytes was analyzed with ratiometric confocal fluorescence measurements. Various P2 receptors activators were tested and compared to the effect of ATP; specifically, UDP, MRS 2768, MRS 2365, bzATP, αβMethylene, 2-meSADP, MRS 4062, and MRS 2768 were analyzed. Antagonists (MRS 2500, 5-BDBD, A438079 and NF449) were tested when 10 μM ATP was applied as the EC₅₀ for ATP activation of the calcium influx in the podocytes was determined to be 10.7 ± 1.5 μM. Several agonists including MRS 2365 and 2-meSADP caused calcium flux. Importantly, only P2Y₁ specific antagonist MRS 2500 (1 nM) precluded the effects of ATP concentrations of the physiological range. Immunohistochemical analysis confirmed that P2Y₁ receptors are highly expressed in the podocytes. We conclude that P2Y₁ receptor signaling is the predominant P2Y purinergic pathway in the glomeruli podocytes and P2Y₁ might be involved in the pathogenesis of glomerular injury and could be a target for treatment of kidney diseases.