To identify optimal hydrogen production conditions using growing cultures of Rhodobacter sphaeroides DSM 158 the effects of varying the reactor’s volumetric power input (0.01–1.4kWm−3) and irradiation intensity (5–2500Wm−2) were investigated in batch and continuous production modes. Irradiation intensity had a greater effect on hydrogen production than volumetric power input. Hydrogen production and photofermentative biomass formation were maximized by irradiation at 2250Wm−2 with a volumetric power input of 0.55kWm−3. The bacterial dry weight (2.64gL−1) and rate of hydrogen production (195mLL−1h−1) achieved under these conditions were greater than any that have previously been reported for batch-mode hydrogen production by R. sphaeroides. Continuous mode experiments (D=0.1h−1) yielded a bacterial dry weight, hydrogen production rate, productivity and hydrogen yield of 2.35±0.18gL−1, 165±6.2mLL−1h−1, 3.96LL−1d−1 and 36.6%, respectively.