The major photosynthetic complexes PSⅠ, PSⅡ, the cytochrome b₆f complex and the ATP synthase complex are located in the thylakoid membrane. Here, the newly developed split-ubiquitin two hybrid system was used to investigate protein-protein interactions of thylakoid membrane proteins. The reaction center D1 protein of PSⅡ, which is encoded by plastid gene psbA, was used as the bait protein, while the D2 encoded by plastid gene psbD, another PSⅡ reaction center protein, and Cytb₆ encoded by plastid gene petB, a component of Cytb6f complex, were used as prey proteins. The yeast two hybrid analysis showed that the D1 protein interacts with D2, but not with the Cytb₆. This result confirms the protein interaction model of thylakoid membrane protein complex, and also means the availability of this system in detecting thylakoid membrane protein interactions. Thus, the split-ubiquitin two hybrid systems could provide an efficient tool to reveal the regulation mechanism of chloroplast proteins biogenesis.