Abstract O2 photoreduction by photosynthetic electron transfer, the Mehler reaction , was observed in all groups of oxygenic photosynthetic organisms [2–4], but the electron transport chain mediating this reaction remains unidentified. We provide the first evidence for the involvement of A-type flavoproteins that reduce O2 directly to water in vitro. Synechocystis sp. strain PCC 6803 mutants defective in flv1 and flv3, encoding A-type flavoproteins, failed to exhibit O2 photoreduction but performed normal photosynthesis and respiration. We show that the light-enhanced O2 uptake was not due to respiration or photorespiration. After dark acclimation, photooxidation of P700 was severely depressed in mutants Δflv1 and Δflv3 but recovered after light activation of CO2 fixation, which gives P700 an additional electron acceptor. Inhibition of CO2 fixation prevented recovery but scarcely affected P700 oxidation in the wild-type, where the Mehler reaction provides an alternative route for electrons. We conclude that the source of electrons for O2 photoreduction is PSI and that the highly conserved A-type flavoproteins Flv1 and Flv3 are essential for this process in vivo. We propose that in cyanobacteria, contrary to eukaryotes, the Mehler reaction produces no reactive oxygen species and may be evolutionarily related to the response of anaerobic bacteria to O2.