We have previously identified the suramin analog 4,4′,4″,4″′-(carbonylbis(imino-5,1,3-benzenetriylbis(carbonylimino)))tetrakis-benzene-1,3-disulfonic acid (NF449) as a low nanomolar potency antagonist of recombinant P2X<sub>1</sub> receptors. Here, we characterize, by two-electrode voltage-clamp electrophysiology, three isomeric suramin analogs designated <i>para</i>-4,4′,4″,4″″-(carbonylbis(imino-5,1,3-benzenetriylbis (carbonylimino)))tetrakis-benzenesulfonic acid (NF110), <i>meta</i>-(3,3′,3″,3″″-(carbonylbis(imino-5,1,3-benzenetriylbis (carbonylimino)))tetra-kis-benzenesulfonic acid (NF448), and <i>ortho</i>-(2,2′,2″,2″″-(carbonylbis(imino-5,1,3-benzenetriylbis (carbonylimino)))tetra-kis-benzenesulfonic acid (MK3) with respect to their potency in antagonizing rat P2X receptor-mediated inward currents in <i>Xenopus laevis</i> oocytes. <i>Meta</i>, <i>para</i>, and <i>ortho</i> refer to the position of the single sulfonic acid group relative to the amide bond linking the four symmetrically oriented benzenesulfonic acid moieties to the central, invariant suramin core. NF448, NF110, and MK3 were >200-fold less potent in blocking P2X<sub>1</sub> receptors than NF449, from which they differ structurally only by having one instead of two sulfonic acid residues per benzene ring. Although the <i>meta</i>- and <i>ortho</i>-isomers retained P2X<sub>1</sub> receptor selectivity, the <i>para</i>-isomer NF110 exhibited a significantly increased activity at P2X<sub>3</sub> receptors (<i>K</i><sub>i</sub> ∼ 36 nM) and displayed the following unique selectivity profile among suramin derivatives: P2X<sub>2+3</sub> = P2X<sub>3</sub> > P2X<sub>1</sub> > P2X<sub>2</sub> >> P2X<sub>4</sub> > P2X<sub>7</sub>. The usefulness of NF110 as a P2X<sub>3</sub> receptor antagonist in native tissues could be demonstrated by showing that NF110 blocks αβ-methylene-ATP-induced currents in rat dorsal root ganglia neurons with similar potency as recombinant rat P2X<sub>3</sub> receptors. Together, these data highlight the importance of both the number and exact location of negatively charged groups for P2X subtype potency and selectivity.