Endothelium-derived vascular relaxing factor (EDRF)¹ is a humoral agent that is released by vascular endothelium and mediates vasodilator responses induced by various substances including acetylcholine and bradykinin². EDRF is very unstable, with a half-life of between 6 (refs 3, 4) and 50 (ref. 5) s, and is clearly distinguishable from prostacyclin⁶. The chemical structure of EDRF is unknown but it has been suggested that it is either a hydroperoxy- or free radical-derivative of arachidonic acid or an unstable aldehyde, ketone or lactone³. We have examined the role of superoxide anion (O₂⁻) in the inactivation of EDRF released from vascular endothelial cells cultured on microcarrier beads and bioassayed using a cascade of superfused aortic smooth muscle strips⁷. With this system, we have now demonstrated that EDRF is protected from breakdown by superoxide dismutase (SOD) and Cu²⁺, but not by catalase, and is inactivated by Fe²⁺. These findings indicate that O₂⁻ contributes significantly to the instability of EDRF.