Hematoxylin, a natural dye commonly used as a histological stain, generates superoxide upon oxidation to its quinonoid product, hematein. The parameters affecting this reaction were assessed in developing a new and versatile assay for superoxide dismutase. The autoxidation of hematoxylin to hematein was accompanied by an increase in absorbance between 400 and 670 nm. The autoxidation rate was proportional to hematoxylin concentration and increased with pH above 6.55. Trace metals accelerated the autoxidation and this effect was eliminated by EDTA. Superoxide dismutase inhibited the autoxidation 90-95% below pH 7.8, but above pH 8.1 the rate was augmented by superoxide dismutase. The rate inhibition at low pH was proportional to the superoxide dismutase concentration up to 70% inhibition. The rate acceleration at high pH was proportional to superoxide dismutase concentration up to approximately 200% acceleration. The autoxidation rate was not significantly affected by ethanol, cyanide, azide, hydrogen peroxide, or catalase. However, the reaction was inhibited by the reducing agents NADH, reduced glutathione, ascorbate, and dithiothreitol, and by undialyzed extracts of Escherichia coli B. When cell extracts were dialyzed prior to assay, the degree of inhibition observed was proportional to the concentration of superoxide dismutase in the extract. These observations form the basis for negative and positive assays of superoxide dismutase which are inexpensive and simple to perform. The negative assay has the added advantage of being applicable at physiological pH.