During physiological stress, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) degradation is accelerated, which is considered to be one of the key factors responsible for photosynthetic decline. A recent study has shown that the large subunit (RbcL) of Rubisco is directly fragmented by hydroxyl radicals in Cucumis sativus leaves under chilling-light conditions. In the present study, we investigated biochemical aspects associated with this in vivo RbcL fragmentation by reactive oxygen species. RbcL fragmentation was observed in C. sativus and Phaseolus vulgaris, but not in Solanum lycopersicum, Glycine max, Oryza sativa, Triticum aestivum, Spinacia oleracea or Arabidopsis thaliana. In C. sativus and P. vulgaris, RbcL fragmentation followed the fragmentation of PsaB, while in the other species, PsaB fragmentation did not occur. In C. sativus and P. vulgaris, the activities of antioxidant enzymes decreased dramatically under chilling-light conditions, and the proportion of uncarbamylated Rubisco increased. These data suggest that in vivo RbcL fragmentation under chilling-light conditions is associated with a combination of events, namely, inactivation of antioxidant enzymes, destruction of photosystem I and an increase of uncarbamylated Rubisco, which can produce hydroxyl radicals via the Fenton reaction at the catalytic site of RbcL.