The effect of tiron (4,5-dihydroxy-1,3-benzene disulfonic acid) on the binding of Ca2+ and its effect on vascular responses of the rat perfused mesenteric bed was studied at concentrations of tiron that are used widely to scavenge superoxide anion. In competition assays in buffered solutions with no tissue present, tiron decreased the fluorescence ratio of fura-FF, a measure of [Ca2+]: the inhibition constant (Ki) of tiron with Ca2+ was 0.692 +/- 0.036 mM. In the mesenteric bed perfused at constant flow and preconstricted with 90 mM KCl, tiron evoked decreases in perfusion pressure of the mesenteric bed in a concentration-dependent manner (Rmax = 43.58 +/- 2.6 mmHg; EC50 = 1.46 +/- 0.33 mM). This vasodilator effect of tiron was similar in the presence of the superoxide anion scavenger, tempol (Rmax = 46.12 +/- 1.87 mmHg; EC50 = 1.34 +/- 0.27 mM). In the presence of 90 mM KCl, increasing concentrations of Ca2+ increased perfusion pressure and tiron shifted the concentration-response curve to Ca2+ to the right. In freshly drawn blood from rats, tiron increased clotting time. The data indicate that tiron binds Ca2+ at concentrations at or below those commonly used to scavenge superoxide anion, an action that may be responsible for a variety of biological responses. The interpretation of effects of tiron in previous work on the role of superoxide anion may need to be re-evaluated.