Alfalfa (Medicago sativa) plants were exposed to drought to examine the involvement of carbon metabolism and oxidative stress in the decline of nitrogenase (N2ase) activity. Exposure of plants to a moderate drought (leaf water potential of -1.3 MPa) had no effect on sucrose synthase (SS) activity, but caused inhibition of N2ase activity (-43%), accumulation of succinate (+36%) and Suc (+58%), and upregulation of genes encoding cytosolic CuZn-superoxide dismutase (SOD), plastid FeSOD, cytosolic glutathione reductase, and bacterial MnSOD and catalases B and C. Intensification of stress (-2.1 MPa) decreased N2ase (-82%) and SS (-30%) activities and increased malate (+40%), succinate (+68%), and Suc (+435). There was also upregulation (mRNA level) of cytosolic ascorbate peroxidase and downregulation of SS, homoglutathione synthetase, and bacterial catalase A. Drought stress did not affect nifH mRNA level or leghemoglobin (Lb) expression, but decreased MoFe- and Fe-proteins. Rewatering of plants led to a partial recovery of the activity (75%) and proteins (>64%) of N2ase, a complete recovery of Suc, and a decrease of malate (-48%) relative to control. The increase in O2 diffusion resistance, the decrease in N2ase-linked respiration and N2ase proteins, the accumulation of respiratory substrates and oxidized lipids and proteins, and the upregulation of antioxidant genes reveal that bacteroids have their respiratory activity impaired and that oxidative stress occurs in nodules under drought conditions prior to any detectable effect on SS or Lb. We conclude that a limitation in metabolic capacity of bacteroids and oxidative damage of cellular components are contributing factors to the inhibition of N2ase activity in alfalfa nodules.This work was supported by Ministerio de Educación y Ciencia (MEC)-Fondos Europeos de Desarrollo Regional (grants nos. AGL2002-02876, AGL2005-01404, and AGL-2005-00274) and by Gobierno de Aragón (group E33). L.N. and R.L. are the recipients of predoctoral fellowships (“Formación de Personal Investigador” program) and J.R. is the recipient of a postdoctoral contract (“Juan de la Cierva” program) from MEC. This work is part of the Ph.D. theses of L.N. (supervised by J.R. and M.B.) and of R.L. (supervised by E.M.G. and C.A.-I.).Peer reviewed