The P2Y(12) receptor plays a crucial role in platelet aggregation. In the present study, we analyzed the properties of non-nucleotide antagonists at the recombinant human P2Y(12) receptor and searched for amino acids involved in the molecular interaction. Receptor function was assessed by measuring the cAMP response element (CRE)-directed luciferase expression in Chinese hamster ovary cells. The cellular cAMP production was accelerated by forskolin; 2-methylthio-ADP was used to activate the wild-type P2Y(12) receptor or mutant constructs. 2-Methylthio-ADP inhibited the CRE-dependent luciferase expression with an IC(50) value of approximately 1 nM. The anthraquinone derivative reactive blue 2 used at increasing concentrations shifted the concentration-response curve of 2-methylthio-ADP to the right in a manner compatible with competitive antagonism (pA(2) value, 7.4). Its analog, 1-amino-4-[4-phenylamino-3-sulfophenylamino]-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate (PSB-0739), showed a markedly higher antagonistic potency with a pA(2) value of 9.8. In cells expressing the R256A-mutant receptor, the potencies of both reactive blue 2 (apparent pK(B), 5.9) and PSB-0739 (apparent pK(B), 9.1) were decreased. The same was true for the pure reactive blue 2 meta- and para-isomers and for the ortho-isomer cibacron blue 3GA. In contrast, the analog, 1-amino-4-[4-anilino-phenylamino]-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate, lacking a sulfonic acid residue at ring D (PSB-0826), showed similar pK(B) values at wild-type (8.4) and R256A-mutant receptors (8.3). In summary, the results demonstrate that PSB-0739 is the most potent competitive non-nucleotide antagonist at the human P2Y(12) receptor described so far. The results also indicate that the sulfonic acid residue at ring D is involved in the interaction of antagonists derived from reactive blue 2 with the residue Arg256 of the human P2Y(12) receptor.