We wished to assess whether the previously developed 3,3-diaminobenzidine (DAB)-Mn" cytochemical method, purportedly specific for superoxide localization, is detecting superoxide 0;-and/or the superoxide product, Oz('Ag). We show here that polymorphonuclear leukocytes (PMNs) produce Oz(' Ag) extracellularly in response to non-phagocytic stimuli and that this production is inhibited by addition of superoxide dismutase, an enzyme typically used to demonstrate that a reaction is mediated by Oi-. Because Oz('Ag) is highly reactive and can be generated from Oi-, the reactivity of a pure chemical source of Oz('Ag) with the cytochemical probe DAB was examined in the presence and absence of Mn**. Reactions between DAB and Oz('Ag), thermally released from 1,4-dimethyl-napthalene-1,4-endoperoxide (DNE), indicated that Oz('Ag) directly reacted with DAB, forming an insoluble DAB polymer, and that this reaction was increased by the presence of Mn++. The direct reaction of Oz('Ag) with DAB was confirmed using near-IR emission spectroscopy. The near-IR emission spectrum of DNE as it was warmed showed the characteristic energy emission peak of OZ('Ag) and the intensity of this peak was reduced by the addition of DAB;