Oxidative stress inhibits the repair of photosystem II (PSII), especially the de novo synthesis of proteins, but does not damage PSII. To investigate the mechanism of the inhibition of protein synthesis by oxidative stress, we constructed an in vitro translation system from cyanobacterium Synechocystis sp. strain PCC 6803 and analyzed the synthesis of the D1 protein under oxidative stress. Cell extracts including thylakoid membranes were incubated with psbA2 mRNA, 14C-labeled Leu, and several compounds required for the translational reaction. The synthesis of the D1 protein was detected in light. This translation system did not require DTT, which is indispensable for in vitro translation systems from wheat germ and tobacco chloroplast, indicating that some reducing compounds accumulated in cyanobacterial extracts. Hydrogen peroxide inhibited the synthesis of the D1 protein, but the inhibition was recovered by addition of catalase, suggesting that the oxidative stress-induced inhibition of translation might be reversible.