The photoproduction of hydrogen by 2-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-inhibited chloroplasts from ascorbate under anaerobic conditions was studied in the pH range 5.0 to 7.5 using methyl viologen (MV), N,N,N',N'-tetramethyl-P-phenylenediamine (TMPD), and excess hydrogenase from Desulfovibrio desulfuricans. (a) At neutral and basic pHs, the photoreduction of MV, which reacted back with photoxidized ascorbate (dehydroascorbate [DHASC]), and the rates of H(2) photoproduction were very low. The slow H(2) photoproduction was explained by the reversible reduction of MV by the photoproduced H(2) (catalyzed by hydrogenase) and its reoxidation by DHASC resulting in H(2) uptake. (b) At pH 5.2, relatively high initial rates of H(2) photoproduction were obtained, which were comparable to the rates of O(2) consumption at pH 5.2 by photosystem I (catalyzed by photoreduced MV). However, accumulation of photoreduced MV under anaerobic conditions was not detected. In the presence of high concentrations of protons, the H(2) uptake by DHASC was very slow because the equilibrium concentration of H(2)-reduced MV was very small, thus allowing H(2) evolution mediated by photoreduced MV to compete with the back reaction with DHASC. (c) The continuous accumulation of DHASC, which was generated together with H(2), gradually slowed the H(2) evolution until it stopped after about 3 hours. At high concentrations, DHASC was able to compete with the coupling of photoreduced MV to hydrogenase and H(2) evolution. (d) Dithiothreitol (DTT) reduced the DHASC and consequently competed with the back reaction of the photoreduced and H(2)-reduced MV with DHASC. DTT thus prolonged the time period of H(2) photoproduction from ascorbate and abolished the dependence of its rate on pH in the range of 5.2 to 7.5 (e) A study of H(2) uptake by chemically oxidized ascorbate (in the dark) showed that MV and hydrogenase were both required to catalyze electron transfer from H(2) to DHASC. TMPD prevented this H(2) consumption by DHASC (in a chloroplast reaction mixture containing MV and hydrogenase). Illumination restored the H(2) uptake presumably by generating reduced MV which activated the hydrogenase.