Summary. The aim of the present study was to characterize the pharmacological profile of the P2Y12 receptor for several adenine triphosphate nucleotides in view of their possible roles as partial agonists or true antagonists. Two distinct cellular systems were used: P2Y1 receptor deficient mouse platelets ( platelets) previously shown to express a native and functional P2Y12 receptor and 1321 N1 astrocytoma cells stably expressing the human P2Y12 receptor (1321 N1 P2Y12). ADP and its structural analogues inhibited cAMP accumulation in a dose-dependent manner in both platelets and 1321 N1 P2Y12 cells with a similar rank order of potency, 2 methylthio-ADP (2MeSADP) >>ADP – Adenosine 5′-(βthio) diphosphate (ΑDPβS). Commercial ATP, 2 chloro; ATP (2ClATP) and 2 methylthio-ATP (2MeSATP) also inhibited cAMP accumulation in both cell systems. In contrast, after creatine phosphate (CP)/creatine phosphokinase (CPK) regeneration, adenine triphosphate nucleotides lost their agonistic effect on platelets and behaved as antagonists of ADP (0.5 µm)-induced adenylyl cyclase inhibition with IC50 of 13.5 ± 4.8, 838 ± 610, 1280 ± 1246 µm for 2MeSATP, ATP and 2ClATP, respectively. In 1321 N1 P2Y12 cells, CP/CPK regenerated ATP and 2ClATP lost their agonistic effect only when CP/CPK was maintained during the cAMP assay. The stable ATP analogue ATPγS antagonized ADPβS-induced inhibition of cAMP accumulation in both platelets and 1321 N1 P2Y12 cells. Thus, ATP and its triphosphate analogues are not agonists but rather antagonists at the P2Y12 receptor expressed in platelets or transfected cells, provided care is taken to remove diphosphate contaminants and to prevent the generation of diphosphate nucleotide derivatives by cell ectonucleotidases.