Nitric oxide (NO) is a bioactive molecule involved in diverse physiological functions in plants. Here we demonstrate that NO is capable of regulating the activity of photophosphorylation in chloroplasts. The electron transport activity in photosystem II determined from chlorophyll a fluorescence was inhibited by NO. NO also inhibited light-induced DeltapH formation across the thylakoid membrane. High concentrations of nitrite and nitrate did not show such inhibitory effects, suggesting that the inhibition is not due to uncoupling effects of the oxidized products of NO. ATP synthesis activity upon illumination was severely inhibited by NO (IC(50)=0.7 microM). The inhibition was found to be temporary and the activity was completely recovered by removing NO. Bovine hemoglobin and bicarbonate were effective in preventing NO-dependent inhibition of photophosphorylation. These results indicate that NO is a reversible inhibitor of photosynthetic ATP synthesis.