Pharmacological properties of the human P2Y1 receptor transfected in Jurkat cells and of the endogenous receptor in rat brain capillary endothelial cells were analyzed under conditions in which the purity of adenine triphosphate nucleotides was controlled by creatine phosphate/creatine phosphokinase (CP/CPK). ATP, a partial agonist of the receptor, was inactive in the presence of CP/CPK. Results further indicated that ATP was a competitive antagonist of ADP actions. Ki values were 23.0 +/- 1.5 microM in endothelial cells and 14.3 +/- 0.3 microM in Jurkat cells. Solutions prepared from commercially available 2-methylthio-ATP (2-MeSATP) or 2-chloro-ATP (2-ClATP) contained approximately 10% of ADP derivatives. ADP derivatives were removed from the solution by treatment with CP/CPK. Purified 2-MeSATP and 2-ClATP antagonized platelet aggregation induced by ADP. They did not activate P2Y1 receptors but prevented ADP actions in a competitive manner. Ki values for 2-MeSATP were 36. 5 microM in endothelial cells and 5.7 +/- 0.4 microM in Jurkat cells, and Ki values for 2-ClATP were 27.5 microM in endothelial cells and 2.3 +/- 0.3 microM in Jurkat cells. EDTA potentiated actions of ADP and ATP on endothelial cells by 2.4- and 3.6-fold, respectively. In conclusion, the rat and human P2Y1 receptors are ADP-specific receptors that recognize ADP and 2-methylthio-ADP, whereas ATP, 2-MeSATP, and 2-ClATP are competitive antagonists. The results further point to the close pharmacological similarity of the P2Y1 receptor and the platelet ADP receptor.