Possible association of photodynamic sensitization by cytochrome b₆/f complex (cyt b₆/f) via singlet oxygen (¹O₂) mechanism with photoinhibition damage to photosystem II (PS II) was studied using such subthylakoid preparations as photosystem I (PS I) particles, PS II core complex and cyt b₆/f from spinach leaves. Upon exposure to bright light, PS II core complex lost photosynthetic electron transport activity to a certain extent, whose spectral dependence implied that pheophytin a is likely involved in photoinactivation of PS II core complex in itself. The presence of PS I particles exerted virtually no effect on PS II core photoinactivation. However, the inclusion of cyt b₆/f in samples resulted in a marked exacerbation of the photoinactivation, particularly in UV-A and blue light. Such effect of cyt b₆/f was suppressed by azide and enhanced by the medium deuteration. Photogeneration of ¹O₂ from cyt b₆/f was confirmed by ESR and spectrophotometry, chemically trapping ¹O₂. Action spectra for both ¹O₂ photoproduction and PS II core photoinactivation by cyt b₆/f bore a close resemblance to each other, seemingly carrying the absorption characteristics of the Rieske Fe-S protein. A complex deficient in the Rieske protein prepared from intact cyt b₆/f showed virtually no generation of ¹O₂ in light, whereas an efficient photoformation of ¹O₂ was seen in the Rieske protein preparation. The results suggest that cyt b₆/f, rather specifically the Rieske center, may play a prominent role in photoinhibition processes through type II photosensitization in thylakoids.