Soluble hydrogenase from a marine green alga, Tetraselmis subcordiformis, was purified to homogeneity and characterized under strict anaerobic conditions. After a 4 h dark, anaerobic adaptation period for the algal culture, the hydrogenase showed a maximum in vitro hydrogen production activity of 148 nmol H2/(μg Chl a × h). The enzyme was purified 362-fold by a three-step process, and using reduced methyl viologen as the electron donor its activity was 61.67 U/mg at 25 °C. The optimum temperature and pH value for hydrogen production were 55 °C and 7.5, respectively. The N-terminal amino acid sequence of this 46 kDa hydrogenase was determined, and it showed no homology to any known hydrogenase sequence.