The production of reactive oxygen species (ROS) was studied in isolated thylakoid membranes exposed to 312 nm UV-B irradiation. Hydroxyl radicals (^•OH) and hydrogen peroxide were measured directly, using a newly developed method based on hydroxylation of terephthalic acid and the homovanillic acid/peroxidase assay, respectively. At the early stage of UV-B stress (doses lower than 2.0 J cm^–2), ^•OH were derived from superoxide radicals via hydrogen peroxide. Production of these ROS was dependent on photosynthetic electron transport and was not exclusive to UV-B. Both ROS were found in samples exposed to the same doses of PAR, suggesting that the observed ROS are by-products of the UV-B-driven electron transport rather than specific initiators of the UV-B-induced damage. After longer exposure of thylakoids to UV-B, leading to the inactivation of PSII centres, a small amount of ^•OH was still observed in thylakoids, even though no free hydrogen peroxide was detected. At this late stage of UV-B stress, ^•OH may also be formed by the direct cleavage of organic peroxides by UV-B. Immunodetection showed that the presence of the observed ROS alone was not sufficient to achieve the degradation of the D1 protein of PSII centres.