This study examines the effect of antimycin A and nitrite on 14CO2 fixation in intact chloroplasts isolated from spinach (Spiucia oeracea L.) leaves. Antimycin A (2 micromolar) strongly inhibited CO2 fixation but did not appear to inhibit or uncouple linear electron transport in intact chloroplasts. The addition of small quantities (40-100 micromolar) of nitrite A stoichiometric balance of ATP/NADPH in the chloroplast is essential for optimum photosynthesis. It is now widely accepted that such a stoichiometric balance of ATP/NADPH could not be achieved by linear electron transport from H20 to NADP+ alone (1 1). Assuming that the ATP/NADPH ratio generated via linear electron transport is 1.33, the extra ATP needed would presumably be derived from cyclic and/or pseudocyclic electron transport.High rates of 02 uptake have been measured in thylakoid preparations from spinach leaves in the presence of high concentrations of Fd (1; R. T. Furbank and M. R. Badger, unpublished), during photosynthesis in intact chloroplasts and cells (8,9), and in leaves of C3 plants (6,10). The values of 02 uptake measured in these studies are comparable to and, in several cases, exceeded the value (1 1.3% of total 02 evolution) required for the generation of all the extra ATP needed for CO2 fixation. This evidence suggests that pseudocyclic electron transport plays a significant (dominant) role in the ATP/NADPH balance during photosynthesis. Marsho et al. (18) found that, although there was a significant rate of 02 uptake in intact chloroplasts during the lag phase of photosynthesis, this diminished to a relatively insignificant level during steady-state photosynthesis. These low rates of 02 uptake were relatively insensitive to increases in light intensity which greatly stimulated photosynthesis (14). These and a number of other workers (3,4,20,23,32) have provided evidence for cyclic flow during photosynthesis in thylakoids, chloroplasts, and intact leaves. Quantitative measurements of Cyt turnover in intact chloroplasts (26) have also indicated substantial cyclic flow. But the corresponding rates of 02 evolution during these measurements were low. Thus, the relative contribution of cyclic versus pseudocyclic flow to the ATP/NADPH balance during photosynthesis remains unresolved. Furthermore, the reasons for the discrepancies between high (8, 9) and low (14, 18) rates of 02 uptake measured during photosynthesis in chloroplasts and cells remain to be resolved.In thylakoids, antimycin A is a reported potent inhibitor of Fddependent cyclic flow (2) but may function as an uncoupler at higher concentrations (7). In intact chloroplasts, antimycin A stimulated CO2 fixation under anaerobic conditions (19,22,25) and in aerobic conditions which were suboptimal (e.g. high Pi concentration) for CO2 fixation (28). But under aerobic conditions at optimal Pi, antimycin A strongly inhibited photosynthetic CO2 fixation in intact chloroplasts with concomitant decreases in energy-dependent processes such as Chl a fluorescence, 9-aminoacridin...