Photoactivation of the latent oxygen-evolving system in intact chloroplasts isolated from wheat (Triticum aestivum L.) leaves grown under intermittent flash illumination was investigated, and the following results were obtained: (a) The water-oxidation activity generated on illuminating the isolated intact chloroplasts was as high as that generated in intact leaves, indicating that all the machinery necessary for the activity generation is assembled within intact chloroplasts. (b) The generation of water-oxidation activity was accompanied by enhancement of the activity of diphenylcarbazide-oxidation, and both processes share the same photochemical reaction but with respective rate-limiting dark reactions of different efficiencies. (c) A23187, an ionophore for divalent cations, strongly inhibited the generation of water-oxidation activity but did not affect the activity once generated, which suggested that Mn atoms in the chloroplasts are susceptible to the ionophore before photoactivation but turn immune after photoactivation. (d) The generation of water-oxidation activity was not affected by the inhibitors of ATP formation and CO2 fixation, but was inhibited by nitrite, methylviologen and phenylmercuric acetate which suppress or inhibit the reduction of ferredoxin in intact chloroplasts. It was inferred that some factor(s) probably present in stroma to be reduced by PSI photoreaction is involved in the process of photoactivation.