The production of superoxide radical (O-2(.-)) was studied in plasma membrane vesicles isolated by aqueous polymer two-phase partitioning from roots of zinc-sufficient and zinc-deficient bean (Phaseolus vulgaris L. cv. Prelude) plants. The two populations of Vesicles were highly enriched in plasma membrane and had similar composition as evidenced by the specific membrane marker enzymes. Vesicles from zinc-deficient roots showed higher rates of NAD(P)H oxidation compared to vesicles from zinc-sufficient plants. The NAD(P)H-dependent formation of O-2(.-) in plasma membrane Vesicles was also highly increased by zinc deficiency. For both activities, a higher response to zinc deficiency was observed when NADPH was used as electron source. Re-supply of zinc to deficient plants for 24 h substantially decreased the rates of NAD(P)H oxidation and O-2(.-) production in isolated vesicles. The NADPH-dependent O-2(.-) generation was strongly stimulated by FAD and showed a high pH optimum; it was scarcely affected by Triton X-100 or even inhibited in the presence of FAD and was almost insensitive to Antimycin A.