Novel analogs of the P2 receptor antagonist pyridoxal-5′-phosphate-6-phenylazo-2′,4′disulfonate (PPADS) were synthesized. Modifications were made through functional group substitution on the sulfophenyl ring and at the phosphate moiety through the inclusion of phosphonates, demonstrating that a phosphate linkage is not required for P2 receptor antagonism. Substituted 6-phenylazo and 6naphthylazo derivatives were also evaluated. Among the 6-phenylazo derivatives, 5′-methyl, ethyl, propyl, vinyl, and allyl phosphonates were included. The compounds were tested as antagonists at turkey erythrocyte and guinea-pig taenia coli P2Y 1 receptors, in guinea-pig vas deferens and bladder P2X 1 receptors, and in ion flux experiments by using recombinant rat P2X 2 receptors expressed in Xenopus oocytes. Competitive binding assay at human P2X 1 receptors in differentiated HL-60 cell membranes was carried out by using [ 35 S]ATP-γ-S. A 2′-chloro-5′-sulfo analog of PPADS (C 14 H 12 O 9 N 3 ClPSNa), a vinyl phosphonate derivative (C 15 H 12 O 11 N 3 PS 2 Na 3 ), and a naphthylazo derivative (C 18 H 14 O 12 N 3 PS 2 Na 2 ), were particularly potent in binding to human P2X 1 receptors. The potencies of phosphate derivatives at P2Y 1 receptors were generally similar to PPADS itself, except for the p-carboxyphenylazo phosphate derivative C 15 H 13 O 8 N 3 PNa and its m-chloro analog C 15 H 12 O 8 N 3 ClPNa, which were selective for P2X vs. P2Y 1 receptors. C 15 H 12 O 8 N 3 ClPNa was very potent at rat P2X 2 receptors with an IC 50 value of 0.82 µM. Among the phosphonate derivatives, [4-formyl-3-hydroxy-2-methyl-6-(2-chloro-5-sulfonylphenylazo)-pyrid-5-yl]methylphosphonic acid (C 14 H 12 -O 8 N 3 ClPSNa) showed high potency at P2Y 1 receptors with an IC 50 of 7.23 µM. The corresponding 2,5-disulfonylphenyl derivative was nearly inactive at turkey erythrocyte P2Y 1 receptors, whereas at recombinant P2X 2 receptors had an IC 50 value of 1.1 µM. An ethyl phosphonate derivative (C 15 H 15 O 11 N 3 PS 2 Na 3 ), whereas inactive at turkey erythrocyte P2Y 1 receptors, was particularly potent at recombinant P2X 2 receptors.