Lucigenin has been widely used as a chemiluminescent substrate to monitor vascular superoxide (O^•−₂) formation. The validity of lucigenin for detection of O^•−₂has been questioned because O^•−₂is generated by lucigenin itself. It has been shown that the concentration of lucigenin is a critical parameter affecting the validity of this assay. In the present studies we evaluated a reduced concentration of lucigenin (5 μM) as a tool to quantify O^•−₂production in vascular tissue. Lucigenin-induced effects on endothelial function were assessed by isometric tension recording of isolated aortic rings suspended in organ baths. The effects of lucigenin on O^•−₂production were studied using spin trapping and electron spin resonance spectroscopy. Lucigenin at 250 μM but not at 5 μM caused a significant attenuation of endothelium-dependent relaxations to acetylcholine, which was prevented by pretreatment with superoxide dismutase. Spin-trapping studies revealed that lucigenin at 250 μM increased vascular O^•−₂production several fold while 5 μM lucigenin did not stimulate O^•−₂production. Inhibition of NO synthase byN^G-momomethyl-l-arginine as well as the removal of the endothelium almost doubled lucigenin-derived chemiluminescence (LDCL), indicating that basal production of endothelium-derived NO depresses the baseline chemiluminescence signal. Thus, lucigenin at a concentration of 5 μM seems to be a sensitive and valid probe for assessing O^•−₂in vascular tissue. It can also be used as an indirect probe to estimate basal vascular NO release.