Whole cells and cell extracts of Eubacterium species V. P. I. 12708 7-dehydroxylated [3H]ursodeoxycholic acid or [14C]chenodeoxycholic forming lithocholic acid. 7 beta-Dehydroxylation specific activity was 146 and 386 nmol hr-1 mg protein-1 for cell extracts and whole cells, respectively. 7 alpha- or 7 beta-Dehydroxylation activity was detected only in whole cells or cell extracts prepared from cultures grown in the presence of cholic acid. The addition of NAD+ (0.5 mM) to anaerobically dialyzed cell extracts stimulated 7 beta- and 7 alpha-dehydroxylation activity by 5- and 40-fold, respectively. The level of 7 beta-dehydroxylation specific activity was approximately 3- to 5-fold lower than 7 alpha-dehydroxylation in whole cells and 3-fold lower in cell extracts. Substrate saturation kinetics for ursodeoxycholic acid and chenodeoxycholic acid were hyperbolic and showed substrate inhibition at concentrations above 200 microM. The apparent Km values for ursodeoxycholic and chenodeoxycholic acid were 14.5 microM and 49 microM, respectively. Both 7 alpha- and 7 beta-dehydroxylase activities were inactivated (60% to 70%) by heating for 6 min at 45 degrees C. Moreover, both activities co-eluted from a anaerobic Bio-Gel A 1.5-M column as a single peak at approximately 114,000 (Mr). These data show that this intestinal anaerobic bacterium has both 7 alpha- and 7 beta-dehydroxylase activities which may be catalyzed by the same enzyme.U