Azido-ioxynil (3.5-diiodo-2-azido-4-hydroxy-benzonitrile) is a potent photosystem II inhibitor (pI50-value 7.38) and as effective as the parent compound ioxynil itself. [125I]azido-ioxynil exhibits specific binding to isolated thylakoids with a binding constant Kb = 7.14. Upon UV-illumination it binds covalently to thylakoids or photosystem II particles. It labels predominantly the 32 kDa D-1 photosystem II reaction center protein. A 41 kDa protein is only tagged in trace amounts. After proteolytic treatment of labeled D-1 protein with Staphylococcus aureus V8-protease two major and two minor fragments are obtained. Automated gas phase sequencing of a 7 kDa cleavage peptide revealed that Val249 is the primary target of azido-ioxynil binding. Compared to urea type herbicides, this places the ioxynil binding site in a different environment of the D-1 photosystem II protein.