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A P2X1-eYFP knock-in mouse was generated to study receptor expression and mobility in smooth muscle and blood cells. eYFP was added to the C-terminus of the P2X1R and replaced the native P2X1R. Fluorescence corresponding to P2X1-eYFPR was detected in urinary bladder smooth muscle, platelets and megakaryocytes. ATP-evoked currents from wild type and P2X1-eYFP isolated urinary bladder smooth muscle cells had the same peak current amplitude and time-course showing that the eYFP addition had no obvious effect on properties. Fluorescence recovery after photobleaching (FRAP) in bladder smooth muscle cells demonstrated that surface P2X1Rs are mobile and their movement is reduced following cholesterol depletion. Compared to the platelet and megakaryocyte, P2X1-eYFP fluorescence was negligible in red blood cells and the majority of smaller marrow cells. The spatial pattern of P2X1-eYFP fluorescence in the megakaryocyte along with FRAP assessment of mobility suggested that P2X1Rs are expressed extensively throughout the membrane invagination system of this cell type. The current study highlights that the spatiotemporal properties of P2X1R expression can be monitored in real time in smooth muscle cells and megakaryocytes/platelets using the eYFP knock-in mouse model. Electronic supplementary material The online version of this article (10.1007/s11302-019-09666-1) contains supplementary material, which is available to authorized users.