Site-directed mutagenesis was used to search for amino acid residues of the human P2Y(1) receptor involved in the binding of the P2 receptor antagonists pyridoxal-5'-phosphate-6-azophenyl-2,4-disulfonate (PPADS), its analogue 6-(2'-chloro-phenylazo)-pyridoxal-α(5)-phosphate (MRS 2210), the suramin analogue 8-8'-[carbonylbis(imino-3,1-phenylene)]bis(1,3,5-naphthalene-trisulfonate) (NF023), and Reactive blue 2. Receptors containing single amino acid replacements at positions in transmembrane helical domains (TMs) 3, 5, 6, and 7 critical for the activation of the receptor by nucleotide agonists were expressed in COS-7 (African green monkey kidney) cells. Inositol phosphate accumulation was induced by 2-methylthioadenosine 5'-diphosphate (2-MeSADP). In wild type human P2Y(1) receptors, PPADS (10 to 60 µM), MRS 2210 (10 µM), NF023 (100 µM), and Reactive blue 2 (10 µM) shifted the concentration-response curve of 2-MeSADP in a parallel manner to the right. For PPADS, a pA(2) value of 5.2 was estimated. The shifts caused by MRS 2210, NF023, and Reactive blue 2 corresponded to apparent pK(B) values of 5.6, 5.0, and 5.8, respectively. In K280A mutant receptors, the affinities of PPADS, MRS 2210, NF023, and Reactive blue 2 were about 6- to 60-fold lower than those observed at wild type receptors. The K280A mutation also caused an approximately 1,000-fold increase in the EC(50) value of the agonist 2-MeSADP, similar to previous observations. In contrast, no major change in antagonistic potency was observed at receptors with other mutations in TMs 3, 5, 6, and 7. Thus, the residue Lys(280) (6.55), which is located within the upper third of TM 6 of the human P2Y(1) receptor, is not only critical for the activation of the receptor but also plays an important role in the binding of pyridoxal derivatives and a number of other chemically unrelated P2 receptor antagonists. Lys(280) seems to belong to an overlapping region of the respective binding sites.