Preparations of plasma membranes isolated from cultured rose (Rosa damascena Mill. cv Gloire de Guilan) cells synthesized O-2(-) when incubated with either NADH or NADPH, as measured by an O-2(-)-specific assay based on the chemiluminescence of lucigenin. The activities were strongly dependent on the presence of Triton X-100. The K-m for NADH was 159 mu M; that for NADPH was 19 mu M. Neither NADH- nor NADPH-dependent activity was inhibited by azide, an inhibitor of peroxidase, nor by antimycin A, an inhibitor of mitochondrial electron transport; both activities were inhibited by 30 to 100 nM diphenylene iodonium, an inhibitor of the mammalian NADPH oxidase. The NADH- and NADPH-dependent activities could be distinguished by detergent solubilization and ultracentrifugation: the NADH-dependent activity sedimented more easily, whereas the NADPH-dependent activity remained in suspension. One or both of these enzymes may provide the O-2(-) seen when plant cells are exposed to pathogens or pathogen-associated elicitors; however, plasma membranes from rose cells treated with a Phytophthora elicitor had the same activity as control cells.