Abstract The effect of enzyme concentration on the H 2-uptake and H 2-evolving activities of the reversible hydrogenase from Thiocapsa roseopersicina was examined. In the activity range assayed by a spectrophotometric technique the apparent H 2-uptake specific activity varied greatly with hydrogenase concentration. Study of H 2-evolving activity measured by the H 2 electrode method and compared with a gas chromatographic assay also indicated that specific activity was highly dependent on enzyme concentration. The results indicate that the widely applied hydrogenase assays give systematically erroneous specific activity values. These assays should be used only for relative measurements and the hydrogenase concentration in the reaction mixture should be kept constant. To make the data from various laboratories comparable the assay parameters should be standardized.