Genes encoding homologs of the gp91^phox subunit of the plasma membrane NADPH oxidase complex have been identified in plants and are hypothesized to be a source of reactive oxygen species during defense responses. However, the direct involvement of the gene products in superoxide (O₂  ⁻) production has yet to be shown. A novel activity gel assay based on protein fractionation in native or sodium dodecyl sulfate (SDS)-denaturing polyacrylamide gels was developed. In native polyacrylamide gel electrophoresis, one or two major O₂  ⁻-producing formazan bands were detected in tomato (Lycopersicum esculentum Mill. cv Moneymaker) and tobacco (Nicotiana tabacum var. Samsun, NN) plasma membranes, respectively. Denaturing fractionation of tomato and tobacco plasma membrane in SDS-polyacrylamide gel electrophoresis, followed by regeneration of the in-gel activity, revealed NADPH-dependent O₂  ⁻-producing formazan bands of 106-, 103-, and 80- to 75-kD molecular masses. The SDS and native activity bands were dependent on NADPH and completely inhibited by diphenylene iodonium or CuZn- O₂  ⁻ dismutase, indicating that the formazan precipitates were due to reduction by O₂  ⁻ radicals catalyzed by an NADPH-dependent flavin containing enzyme. The source of the plasma membrane activity bands was confirmed by their cross-reaction with antibody prepared from the C terminus of the tomato gp91^phoxhomolog. Membrane extracts as well as the in-gel NADPH oxidase activities were stimulated in the presence of Ca²⁺. In addition, the relative activity of the gp91^phox homolog was enhanced in the plasma membrane of tobacco mosaic virus-infected leaves. Thus, in contrast to the mammalian gp91^phox, the plant homolog can produce O₂  ⁻ in the absence of additional cytosolic components and is stimulated directly by Ca²⁺.