1. The symmetrical 3'-urea of 8-(benzamido)naphthalene-1,3,5-trisulfonic acid (NF023) and pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) were investigated for their ability to antagonize responses mediated via P2-purinoceptors. In addition, putative inhibitory effects of these compounds on ecto-nucleotidase activity were studied. 2. In rabbit vas deferens, PPADS antagonized contractions to alpha, beta-methylene ATP (alpha, beta-mATP) in a pseudoirreversible manner (pA2 = 6.34). This P2X-antagonism by PPADS has been confirmed in certain vascular and visceral smooth muscles (pA2 = 6.02-6.41). PPADS inhibited P2Y-receptor-mediated relaxant responses to ADP-beta-S or 2-methylthio ATP (2-MeSATP) in guinea-pig taenia coli (pA2 = 4.59 and 5.26, respectively) as well as in rat duodenum (pA2 = 5.09) and mesenteric artery (pA2 = 5.46). Experiments on the rat mesenteric arterial bed demonstrated the ineffectiveness of PPADS at P2U-purinoceptors. P2T-purinoceptor-mediated platelet aggregation was affected only at high concentrations of PPADS (> 100 microM). PPADS (100 microM) was also weakly active in inhibiting ecto-nucleotidase activity in guinea-pig taenia coli. 3. In rabbit vas deferens, NF023 antagonized P2X-purinoceptor-mediated contractions to alpha, beta-mATP in a competitive manner. The estimated pA2 value of 5.68 was very similar to that obtained at P2X-receptors in rat mesenteric (pA2' = 5.54) and rabbit saphenous artery (pA2 = 5.69). In rat duodenum and guinea-pig taenia coli, NF023 competitively inhibited relaxant responses to the P2Y-selective agonists, ADP-beta-S and 2-MeSATP (pA2 = 4.00-4.25). In rat mesenteric arterial bed, NF023 antagonized vasodilator responses mediated by P2Y-purinoceptors (pA2 = 4.94), but had no effect on vasodilator responses to the P2U-selective agonist, UTP. NF023 was found to inhibit ectonucleotidase activity in guinea-pig taenia coli and rabbit vas deferens (pIC40 = 3.52 and 4.12, respectively). 4. At 100 microM, PPADS and NF023 did not interact with alpha 1- and alpha 2-adrenoceptors, adenosine A1- and A2-, histamine H1- and muscarinic M1-, M2- and M3-receptors. 5. In conclusion, the results demonstrate that PPADS and NF023 are specific P2-receptor antagonists and useful tools in the study of multiple subtypes of P2-purinoceptors.