Abstract  Platelet Derived Growth Factor Receptor α positive (PDGFRα+) cells are suggested to mediate purinergic inputs in GI muscles, but responsiveness of these cells to purines in situ has not been evaluated. We developed techniques to label and visualize PDGFRα+ cells in murine gastric fundus, load cells with Ca2+ indicators, and follow their activity via digital imaging. Immuno‐labeling demonstrated a high density of PDGFRα+ cells in the fundus. Cells were isolated and purified by FACS using endogenous expression of eGFP driven off the Pdgfra promoter. Quantitative PCR showed high levels of expression of P2Y1 receptors and SK3 channels in PDGFRα+ cells. Ca2+ imaging was used to characterize spontaneous Ca2+ transients and responses to purines in PDGFRα+ cells in situ. ATP, ADP, UTP and β‐NAD elicited robust Ca2+ transients in PDGFRα+ cells. Ca2+ transients were also elicited by the P2Y1‐specific agonist N)‐methanocarba‐2MeSADP (MRS‐2365), and inhibited by MRS‐2500, a P2Y1‐specific antagonist. Responses to ADP, MRS‐2365 and β‐NAD were absent in PDGFRα+ cells from P2ry1(‐/‐) mice, but responses to ATP were retained. Purine evoked Ca2+ transients were mediated through Ca2+ release mechanisms. Inhibitors of PLC (U‐73122), IP3 and ryanodine receptors, SERCA pump (cyclopiazonic acid (CPA) and thapsigargin) abolished Ca2+ transients elicited by purines. This study provides a link between purine binding to P2Y1 receptors and activation of SK3 channels in PDGFRα+ cells. Activation of Ca2+ release is likely to be the signaling mechanism in PDGFRα+ cells responsible for transduction of purinergic enteric inhibitory input in gastric fundus muscles. This article is protected by copyright. All rights reserved