Assay of hydrogenase activity pertaining to H-2 production needs anaerobic conditions. To establish a simplified method for assay of hydrogenase activities by using intact cells of Enterobater aerogenes, different chemicals capable of enhancing the cell-wall permeability to electron mediators were examined. As a result, Triton X-100 and CTAB were found to be appropriate for H-2 uptake and evolution activities of the intact cells, respectively. This method enabled H-2 uptake and evolution activities of the intact cells to be easily detected. This is also the first report of the presence of H-2 uptake hydrogenase activity in E. aerogenes.