Spinach chloroplast preparations were mixed with Clostridium kluyveri hydrogenase and ferredoxin. Hydrogen evolution could be measured in the light in the absence of any added electron donors. Inhibition of the water-splitting reaction or of photosystem II reduced the amount of H(2) evolved more than 95%, indicating that H(2)O was the electron donor in this reaction. The rates of H(2) evolution observed were up to 20% of those measured in the presence of an oxygen-consuming reaction or of photosystem I electron donors. These findings indicate that hydrogen evolution from water and sunlight by photosynthetic processes could be a method for solar energy conversion.