Rhodobacter sphaeroides RV cultivation and hydrogen production were studied in a one- and two-stage chemostat using lactic acid as substrate. Light saturation was observed when light intensities equal to or above 10 klx were applied. Under light saturation, the two-stage chemostat appeared to be very effective for hydrogen production, allowing complete nitrogen removal by bacterial growth in the first reactor, The hydrogen evolution rate in the second reactor was up to 75 ml H-2 (g dry weight)(-1) h(-1). Accumulation of storage material was observed in the second reactor of the two-stage chemostat under a large carbon excess and limiting light irradiance. The optimal hydraulic residence time was 15 h for both stages, leading to a total hydrogen production about 40% higher than in the one-stage chemostat, Under increasing in fluent ammonium and yeast extract concentrations, opposite trends of decreasing bacterial activity and increasing concentration resulted in a linear increase of the overall hydrogen production to 1.4-1.6 1 H-2 (1 reactor)(-1) day(-1). Hydrogen production quickly fell when nitrogen was not completely metabolised. The hydrogen evolution rate was also found to depend on lactic acid concentration, and maximum bacterial activity was observed at 100 mM influent lactic acid.