Hydrogen production by suspended culture, entrapped culture of Rhodobacter sphaeroides and Clostridium butyricum, and entrapped co-culture of these two bacteria in the presence of NH4+ was investigated and compared. Hydrogen production by suspended culture of Rhottobacter sphaeroides was most affected by NH4+ among the four cultures studied. Nevertheless, hydrogen production rate was the highest after the NH4+ was removed metabolically. Hydrogen production by entrapped culture of Rb. sphaeroides was not readily inhibited by NH4+ compared with that by suspended culture, and the hydrogen production rate was also kept high. Hydrogen production by entrapped culture of C butyricum was not inhibited by NH4+ in the initial period of time, but NH4+ had some influence on hydrogen production by this culture in the later period. The hydrogen production rate from this culture was low compared with that from the culture of Rb. sphaeroides as expected theoretically. The former was only 1 of 5.6 of the latter, on average. The pattern of NH4+ effect on the hydrogen production by co-culture of these two bacteria was similar to that by the single culture of C butyricum. Nevertheless, hydrogen production by co-culture was enhanced by 160% compared with that by the single culture of anaerobic bacterium.