Studies of nucleotide receptors (P2‐receptors) in cells and tissues are complicated by cleavage of phosphate groups from nucleotide agonist ligands by ecto‐nucleotidases. Some P2 receptor antagonists may also inhibit ecto‐nucleotidases, making these studies even more complex. In order to systematically approach this problem, we investigated structure–activity relationships of pyridoxal‐5′‐phosphate‐6‐azophenyl‐2,4‐disulfonate (PPADS) and 14 derivatives, many potent as antagonists at P2 receptors, as inhibitors of ecto‐nucleotidases. The compounds were tested for their ability to inhibit enzymatic nucleotide breakdown by CHO cells stably transfected with plasmids containing the cDNA for rat ecto‐apyrase (NTPDase1) and rat ecto‐ATPase (NTPDase2). All inhibitors were tested at a concentration of 100 μM and ATP hydrolysis was quantified by HPLC. Maximal inhibition obtained for ecto‐apyrase and ecto‐ATPase was 60% and 35%, respectively. Most PPADS analogs were better inhibitors of ecto‐apyrase than of ecto‐ATPase. Compound 8, a phosphate derivative, inhibited ecto‐apyrase with no inhibition evident at ecto‐ATPase. Comparison of pharmacological data of PPADS analogs at P2 receptors as previously determined showed that four PPADS analogs exhibited selectivity for P2X nucleotide receptors. None of these compounds inhibited ecto‐ATPase, while two inhibited the ecto‐apyrase. Compound 14, a bisphosphate derivative, inhibited ecto‐ATPase without inhibition of ecto‐apyrase. This compound only weakly antagonized P2X1 receptors and was inactive at P2X2 and P2Y1 receptors, thus bearing some selectivity for ecto‐ATPase. Compound 7, a 5‐methylphosphonate derivative, a potent antagonist of P2X1 receptors, was inactive at ecto‐apyrase and only weakly inhibitory at ecto‐ATPase. Thus, PPADS modifications that enhance selectivity among ecto‐nucleotidases and P2 receptors have been identified. Drug Dev. Res. 51:153–158, 2000. Published 2001 Wiley‐Liss, Inc.