Common beans (Phaseolus vulgaris L.) were exposed to continuous darkness to induce nodule senescence, and several nodule parameters were investigated to identify factors that may be involved in the initial loss of N2 fixation. After only 1 d of darkness, total root respiration decreased by 76% and in vivo nitrogenase (N2ase) activity decreased by 95%. This decline coincided with the almost complete depletion (97%) of sucrose and fructose in nodules. At this stage, the O2 concentration in the infected zone increased to 1%, which may be sufficient to inactivate N2ase; however, key enzymes of carbon and nitrogen metabolism were still active. After 2 d of dark stress there was a significant decrease in the level of N2ase proteins and in the activities of enzymes involved in carbon and nitrogen assimilation. However, the general collapse of nodule metabolism occurred only after 4 d of stress, with a large decline in leghemoglobin and antioxidants. At this final senescent stage, there was an accumulation of oxidatively modified proteins. This oxidative stress may have originated from the decrease in antioxidant defenses and from the Fe-catalyzed generation of activated oxygen due to the increased availability of catalytic Fe and O2 in the infected region.This work was supported by grant no. PB95-O091 from the Dirección General de Enseiíanza Superior (Spain) to M.B. Work at the Institute of Grassland and Environmental Research was funded through the Biotechnology and Biological Sciences Re- search Council (UK). Y.G., P.R.E., and J.F.M. were the recipients, respectively, of a postdoctoral contract, a predoctoral fellowship, and a postdoctoral fellowship from the Ministerio de Educación y Cultura (Spain).Peer reviewed