A fluorescent photoreactive ATP derivative, 2′(3′)-O-(4-benzoylbenzoyl)-1,N6-etheno-ATP (Bz2-εATP), was synthesized and reacted with the rice kinesin K16 motor domain (K16MD). In the presence of ADP or ATP, UV irradiation of the K16MD solution containing Bz2-εATP resulted in a new 100 kDa band, which was an intermolecular cross-linked product of motor domains. In contrast, no cross-linking was observed in the absence of nucleotides. For the motor domain of mouse brain kinesin and skeletal muscle myosin subfragment-1, no such intermolecular photo cross-linking by Bz2-εATP was observed. Our results indicate that Bz2-εATP acts unusually as a photoreactive crosslinker to detect conformational changes in K16MD induced by nucleotide binding resulting in the formation of dimers.