The generation of active oxygen species by microsomes isolated from soybean seedlings was studied. NADPH-dependent superoxide anion production was 5.0 ± 0.4 nmol · min−1 mg−1 of microsomal protein. Hydrogen peroxide generation by microsomes was 1.40 ± 0.05 nmol · min−1 mg−1 of protein. Hydroxyl radical production, in the presence of ferric EDTA, evaluated through the generation of formaldehyde from dimethyl sulfoxide or tert-butyl alcohol was 0.50 ± 0.04 and 0.44 ± 0.03 nmol · min−1 mg−1, respectively. NADH proved to be suitable as cofactor for oxygen radical generation by microsomes from soybean seedlings. Because transition metals are implicated in radical generation by biological systems, the ability of microsomal membranes to reduce iron complexes was studied. Ferric ATP, ferric citrate, ferric ADP, ferric diethylenetriamine pentaacetic acid, and ferric EDTA were efficiently reduced in the presence of either NADPH or NADH as cofactor. The pattern of effectiveness of the different ferric complexes, on superoxide anion, hydrogen peroxide, and hydroxyl radical production, was similar to that found with animal microsomes. The data presented here indicate that microsomal ability to catalyze oxygen radical generation must be considered as an important contribution to cellular radical steady-state concentrations in cells from soybean seedlings.