In the present study, using specific antibody against D1 protein, we detected four aggregates of D1 protein in thylakoid membranes from spinach leaves illuminated at high light (800―2500 μmol photons·m-2·s-1) for 3 h. Their accumulations were dependent on the light intensity to which the leaves had been subjected. Further immunoblot analysis indicated that 70 kD aggregate was a product of D1 protein cross-linked with CP43, 65 and 60 kD aggregate were two cross-linked products between D1 and D2 proteins, and 41 kD aggregate was one cross-linked D1 with α-subunit of cytochrome b559 (Cyt b559). This result provided the evidence for the existence of the aggregation of the D1 protein in vivo. The maximal level of D1/Cyt b559 aggregate occurred at 1000 μmol photons·m-2·s-1 but drastically decreased with further increasing light intensity. Immunoblot analysis with phosphothreonine (Thr (P)) antibody indicated that D1/CP43 and D1/Cyt b559 aggregates contained the phosphorylated protein(s). In vitro dephosphorylation experiment also showed that D1/Cyt b559 and D1/CP43 aggregates lost the immunoreactivity with Thr (P) antibody after the phosphatase treatment of the membranes from high-light-illuminated leaves. Our results demon- strated that strong illumination of spinach leaves induced cross-linked products of D1 protein with its nearby polypeptides of PS Ⅱ, some of which contained the phosphorylated D1 protein.