ADP a critical regulator of platelet activation, mediates its actions through two G protein-coupled receptors (GPCRs), the P2Y1 and P2Y12 purinergic. Recently we demonstrated that both receptors desensitize (1) and internalise (2-3) in human platelets by differential kinase-dependent mechanisms. In this study we examined if these responses resensitized following agonist removal and if receptor trafficking played a significant role in this process. All experiments were undertaken in human platelets or in 1321N1 cells stably transfected with either the P2Y1 or P2Y12 receptor. P2Y1 Gq-coupled and P2Y12 Gi-coupled receptor activity was assessed as previously described (1). Receptor surface loss was assessed in 1321N1 cells by ELISA whilst in parallel experiments changes in P2Y1 and P2Y12 surface receptor levels were measured in fixed platelets using the P2Y radioligand [3H] 2MeSADP in combination with the P2Y1 receptor antagonist A3P5P and the P2Y12 receptor antagonist AR-C69931MX (2-3). Initial experiments showed that responses to both of these receptors rapidly resensitize following agonist-dependent desensitisation in human platelets or in 1321N1 cells. Further we discovered that in both systems following agonist-induced internalization both the P2Y1 and P2Y12 receptor rapidly recycle to the cell surface. In 1321N1 cells expression of a dynamin dominant-negative mutant (K44A) which blocks P2Y1 and P2Y12 receptor internalization or treatment with either the phosphatase inhibitor okadoic acid or with monensin which inhibits receptor recycling attenuated receptor resensitization. Further in human platelets treatment with either monensin or okadoic acid significantly attenuated resensitization of both P2Y1 and P2Y12 purinergic receptor responses. In conclusion this study is the first to show that both P2Y1 and P2Y12 receptor activity is rapidly and reversibly modulated in human platelets and reveals that receptor trafficking is an essential part of this process.