The fluorogenic oxidation of hydroethidine (HE) to ethidium (E⁺) has been used as a measure of O₂⁻. Evaluation of this method confirms that O₂⁻, but not O₂ or H₂O₂, rapidly oxidizes HE to E⁺. However the ratio of E⁺ produced per O₂⁻ introduced decreased as the flux of O₂⁻ was increased. This suggested that HE can catalyze the dismutation of O₂⁻ and this was affirmed. HE was oxidized to a red product, distinct from E⁺ by ferricytochrome c and a similar oxidation may occur within Escherichia coli. HE inhibited the growth and killed a SOD-null strain to a greater extent than the SOD-replete parental strain and these effects were much diminished under anaerobic conditions. This indicated that E⁺ was responsible for the toxicity of HE and indeed E⁺ was seen to be toxic under both aerobic and anaerobic conditions. In view of the data presented HE can be recommended as a qualitative but not as a quantitative measure of O₂⁻.