We used horseradish peroxidase and either scopoletin, homovanillic acid, or phenol red to measure hydrogen peroxide generated by human neutrophils. With these assays, superoxide dismutase significantly increased the amount of hydrogen peroxide detected. In contrast, it had no effect when the accumulation of hydrogen peroxide was measured with a hydrogen peroxide electrode. We propose that superoxide interferes with horseradish peroxidase-dependent assays so that hydrogen peroxide is underestimated. Thus, when using these assays, superoxide dismutase must be added to neutrophils to ensure that all the hydrogen peroxide they produce is detected.