The effect of glucose and the uncoupler Cl-CCP upon hydrogen production was studied in adapted cells of Scenedesmus obliquus D3. Cl-CCP at 10(-5)M concentration completely inhibited the evolution of H2 in the dark and increased the apparent rate of H2 evolution in the light. At 10(-5)M Cl-CCP, photosynthesis and photoreduction by anaerobically adapted algae were only temporarily inhibited; O2 evolution reappeared after approximately 1 hr of illumination if CO2 was present. Increasing the Cl-CCP concentration to 5 x 10(-5)M led to a maximum rate of photohydrogen production and fully inhibited H2 evolution, photoreduction and dark H2 evolution. H2 evolution was accompanied by a release of varying amounts of CO2 in the light, as well as in the dark. Dark CO2 production was stimulated by Cl-CCP. H2 evolution in the light was stimulated by adding glucose to autotrophically grown cells or by growing the cells heterotrophically with glucose; starvation had an opposite effect. Adapted cells released (14)CO2 from the 3 and/or 4 position of specifically labeled glucose, indicating that degradation occurred via the Embden-Meyerhof pathway. The amount of H2 released by autotrophically grown cells was the same either with continuous illumination or with short periods of light, followed by darkness. Scenedesmus mutant No. 11, which is unable to evolve O2 was not inhibited in its capacity to evolve H2 in the light. These data indicate that the evolution of H2 in the light by adapted Scenedesmus depends upon the degradation of organic material and does not require the production of free O2 by photosystem II.