Using the tetrazolium salt XTT (Na,3′-[(phenylamino)-carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzenesulfonic acid hydrate) as a sensitive and physiologically compatible probe for the determination of superoxide (O₂ ^·−) production in vivo, we have shown that maize (Zea mays L.) coleoptiles possess the capacity of generating O₂ ^·− in the apoplastic space. Our results are in agreement with the notion that this activity is localized at the plasma membrane and can be attributed to an O₂ ^·−-synthesizing enzyme with catalytic and kinetic properties similar to that of the NADPH oxidase of mammalian phagocytes, with the important exception that it utilizes NADH instead of NADPH as electron donor. When applied to the apoplastic space, NADH strongly increased the O₂ ^·−-producing activity of coleoptiles. The maize NADH-dependent O₂ ^·−-synthase activity could clearly be differentiated from peroxidase-mediated O₂ ^·−-synthesizing activity by its insensitivity to cyanide and azide, as well as by its much higher affinity to O₂. Formation of O₂ ^·−, and concomitantly appearing H₂O₂, was preferentially localized in the outer epidermis of the coleoptile. The physiological significance of O₂ ^·− and H₂O₂ production in relation to the growth-controlling function of the epidermal cell wall is discussed.