An Ndh-deficient mutant of tobacco (Nicotiana tabacum cv. Petit Havana) was prepared by disrupting the ndhF gene in a transplastomic approach. The mutant (ΔndhF) showed 10% of the Ndh complex activity (EC 22.214.171.124) and 8% of the NDH-F polypeptide of that of non-transformed plants. However, in ΔndhF, NDH-A, another Ndh polypeptide, was still present at 50% of the level in non-transformed plants. ΔndhF tobacco showed higher sensitivity than non-transformed plants to photo-oxidative stress (as judged by chlorophyll bleaching) caused by increased light intensity and paraquat applications. These photo-oxidative treatments increased the amount and activity of the Ndh complex, thylakoid peroxidase, post-illumination chlorophyll fluorescence and non-photochemical quenching (NPQ) of chlorophyll fluorescence in non-transformed but not in ΔndhF tobacco. Highly stressed non-transformed plants showed a rapid post-rise decline of chlorophyll fluorescence, probably indicating a re-oxidation of reduced plastoquinone. The results indicate that, in normal plants, the Ndh complex and thylakoid peroxidase (EC 126.96.36.199) provide and remove electrons, respectively, to balance the redox level of the intermediates of cyclic electron transport. In this way, they optimize the generation of the transmembrane H+ gradient of thylakoids and, as a consequence, increase the NPQ and the protection against photo-oxidative stress.