Abstract : To investigate the role of mitochondria in drought stress, the response to water deprivation was compared between Nicotiana sylvestris wild type (WT) plants and the CMSII respiratory complex I mutant. In CMSII, alternative NAD(P)H-dehydrogenases bypassing complex 1 allow respiration.. The difference of mitochondrial function between WT and CMSII plants affect also photosynthesis. The CMSII has lower photosynthetic actitvity than the WT and lower stomatal (gs) and internal (gm) conductances to CO2. When watering of plants with similar leaf surface and similar shoot/root ratio was stopped the relative water content (RWC) declined faster in WT as compared to CMSII leaves. Furthermore, CMSII and WT leaves had the same osmotic potential at leaf saturation (P0) and at leaf turgor lost pressure (PTLP) and the same stomatal density. The slower decline of RWC in CMSII, compared to WT leaves, was most likely the consequence of the lower stomatal conductance (gs) under well-watered conditions and during the first days after withholding watering, The lower stomatal conductance of well-watered CMSII leaves correlated with a lower hydraulic conductance of leaves. Remarkably, total free amino acid levels declined and total soluble protein content increased in CMSII leaves, while the opposite was observed in WT leaves. This suggests protein synthesis in CMSII but protein degradation in WT leaves during drought stress. We also show that CMSII leaves better acclimate to drought stress than the WT leaves. After several days at 80 % RWC , photosynthesis is higher in the mutant than in WT. As compared to the WT, the mutant shows higher rates of photorespiration before and after acclimation to drought.The strong accumulation of glycine in the WT suggests that photorespiration may be limited at the level of glycine decarboxylase. In addition, after acclimation to drought gm declined markedly in WT but not in CMSII leaves, thus further limiting CO2 supply for photosynthesis in the WT. The resulting lower photosynthesis and photorespiration in WT leaves affect also the primary reaction of photosynthesis by increasing the non-photochemical fluorescence quenching (NPQ) and decreasing linear electron transport.