In isolated intact chloroplasts, maximal rates of photosynthetic O2 evolution (in saturating HCO3) are associated with a critical transthylakoid proton gradient as a result of the stoichiometric consumption of 2 mol NADPH and 3 mol ATP/mol CO2 fixed. Studies with the fluorescent probe 9-aminoacridine reveal that in the illuminated steady state the critical delta pH is 3.9. CO2-dependent O2 evolution is inhibited by increases of 0.1-0.2 in delta pH that occur when catalase is omitted from the medium, NO2- is included as an electron acceptor, or when chloroplasts are illuminated under low partial pressures of O2. Low concentrations of antimycin (0.33 microM) or NH4Cl (0.33 mM)decrease delta pH and relieve this inhibition of electron flow. The energy transfer inhibitor quercetin lowers the high ATP/ADP ratio associated with these conditions, but does not lower delta pH or relieve the inhibition. A decrease of delta pH below 3.9 by weaker illumination, millimolar levels of NH4Cl or micromolar levels of antimycin, results in lower rates of photosynthesis owing to limitation by the phosphorylation rate. These findings show that in absence of rate limitation by the carbon cycle, the extent of thylakoid energization is related to the ratio of ATP to NADPH production and in turn, the rate of CO2 assimilation.