H2 production was studied in the following rumen anaerobes: Bacteroides clostridiiformis, Butyrivibrio fibrisolvens, Eubacterium limosum, Fusobacterium necrophorum, Megasphaera elsdenii,Ruminococcus albus and R. flavefaciens. Clostridium pasteurianum and Escherichia coli were included for comparative purposes. H2 production from dithionite, dithionite-reduced methyl viologen, pyruvate and formate was determined. All species tested produced H2 from dithionite-reduced methyl viologen, but only C. pasteurianum, B. clostridiiformis, E. limosum and M. elsdenii produced H2 from dithionite. All species except E. coli produced H2 from pyruvate, but activity was low or absent in extracts of E. limosum, F. necrophorum, R. albus and R. flavefaciens unless methyl viologen was added. H2 was produced from formate only by E. coli, B. clostridiiformis, E. linosum, F. necrophorum and R. flavefaciens. Extracts were subjected to ultracentrifugation in an effort to determine the solubility of hydrogenase. The hydrogenase of all species except E. coli appeared to be soluble, although variable amounts of hydrogenase activity were detected in the pellet. Treatment of extracts of the rumen microbial species with DEAE-cellulose resulted in loss of H2 production from pyruvate. Activity was restored by the addition of methyl viologen. H2 production in these rumen microorganisms is apparently similar to that in the saccharolytic clostridia.