The plasma membrane bound nucleoside triphosphate diphosphohydrolase (NTPDase)-1, 2, 3 and 8 are major ectonucleotidases that modulate P2 receptor signaling by controlling nucleotides' concentrations at the cell surface. In this report, we systematically evaluated the effect of the commonly used P2 receptor antagonists reactive blue 2, suramin, NF279, NF449 and MRS2179, on recombinant human and mouse NTPDase1, 2, 3 and 8. Enzymatic reactions were performed in a Tris/calcium buffer, commonly used to evaluate NTPDase activity, and in a more physiological Ringer modified buffer. Although there were some minor variations, there were no major changes either in the enzymatic activity or in the profile of NTPDase inhibition between the two buffers. Except for MRS2179, all other antagonists significantly inhibited these ecto-ATPases; NTPDase3 being the most sensitive to inhibition and NTPDase8 the most resistant. Estimated IC(50) showed that human NTPDases were generally more sensitive to the P2 receptor antagonists tested than the corresponding mouse isoforms. NF279 and reactive blue 2 were the most potent inhibitors of NTPDases which almost completely abrogated their activity at the concentration of 100 microM. In conclusion, reactive blue 2, suramin, NF279 and NF449, at the concentrations commonly used to antagonize P2 receptors, inhibit the four major ecto-ATPases. This information may reveal useful for the interpretation of some pharmacological studies of P2 receptors. In addition, NF279 is a most potent non-selective NTPDase inhibitor. Although P2 receptor antagonists do not display a strict selectivity toward NTPDases, their IC(50) values may help to discriminate some of these enzymes.