The O(2) concentration in intact and osmotically disrupted isolated spinach (Spinacia oleracea, L.) chloroplasts during photosynthesis was estimated. The chloroplasts were allowed to reduce 3-phosphoglycerate, CO(2), or ferricyanide in light until the rate of O(2) production was linear. When the light was turned off O(2) evolution from the chloroplasts continued for a few seconds. This prolonged O(2) evolution is due to an O(2) surplus inside the chloroplasts which equilibrates with that in the medium. From this surplus the O(2) concentration inside the chloroplasts at the moment when the light had been switched off was calculated. In all experiments the O(2) concentration inside the photosynthesizing chloroplasts was higher than that outside, but was dependent upon the O(2) concentration of the chloroplast medium. At low external O(2) concentration (30 mum) the ratio of the internal to the external O(2) concentration was about 5, whereas at concentrations corresponding to those in airsaturated water this ratio was close to 1. With osmotically broken chloroplasts this ratio was 1.2 at 30 mum O(2) and almost 1 from 150 mum onward. When the O(2) surplus found in broken chloroplasts during photosynthesis was related to the volume of the thylakoids, a ratio of about 2.3 was observed.