The aim of this work was to characterize the role of nitric oxide and hydrogen peroxide during the development and senescence of soybean cotyledons. Soybean cotyledons showed an initial increase in water content, followed by constant fresh weight of the cotyledons up to day 16 of growth, and a significant decay afterwards. A dramatic reduction in dry weight was observed starting at day 7 of growth. Both nitric oxide (NO) content and nitrite-dependent NO generation assessed by electron paramagnetic resonance (EPR) showed a maximum at day 6 of growth. H₂O₂ generation by intact cotyledons was assessed employing the oxidation of 2´-7´-dichlorofluorescin to a fluorescent product. H₂O₂ generation remained relatively stable from day 5 to 12 after germination, showing a significant increase from day 14 to 22 after germination. NO could be related to the mechanism which assures the optimal functionality of the cotyledons during the initial phase after germination started. Meanwhile, reactive oxygen species, probably H₂O₂, could be a part of a complex signaling pathway that triggers the starting of senescence and further falling of the cotyledons.