New media and methods were used in attempts to isolate cholesterol-reducing bacteria (CRB) that might be used to reduce the cholesterol contents of foods. Media for the enrichment and isolation of CRB were formulated, and a variety of novel sources were sampled to isolate CRB with improved properties;Stable mixed cultures of anaerobic bacteria that converted cholesterol to coprostanol were obtained from the following sources: feces from cattle, goats, horses, and humans; a marine sponge; sediments from rivers, streams, and ponds; standing water; a hog sewage lagoon; and commercial frozen pork brains. Cholesterol-reducing activity was maintained in these cultures after numerous transfers in a cholesterol- and lecithin-containing medium;A new anaerobic plating medium was used to successfully isolate pure cultures of CRB from samples obtained from a hog sewage lagoon and standing water. The hog sewage lagoon isolate was characterized and has been designated Eubacterium sp. strain HL. This bacterium converted approximately 90% of the cholesterol in the growth medium to coprostanol after 2 d of incubation. Strain HL is a small, Gram-positive, anaerobic, nonsporing rod. Lecithin, but not cholesterol, was required for growth, and the strain possessed phospholipase activity. Coprostanol was not produced when phosphatidyl inositol or phosphatidyl glycerol was substituted for phosphatidyl choline;Strain HL did not reduce nitrate, produce indole, or hydrolyze starch or gelatin. Esculin was hydrolyzed. Much acid was produced by the fermentation of amygdalin, lactose, and salicin. Strain HL weakly fermented arabinose, cellobiose, fructose, glucose, mannose, and melibiose. Acetic, formic, and succinic acids were produced. The percentages of headspace gas volumes occupied by H[subscript]2 and CO[subscript]2 ranged from 4.5 to 7.2 and 0.9 to 1.8, respectively. Eubacterium strain HL survived exposure to air for up to 48 h;A resting-cell assay was developed, so that a variety of parameters that affected growth and enzyme activity could be investigated. Radiolabeled cholesterol was used as the substrate. Media and cultural methods were then optimized. Optimal activity was achieved after incubation of strain HL at 37°C for 24 to 48 h in a growth medium that was supplemented with 0.5% sodium pyruvate and 0.5% lactose. Pyruvate also stimulated cholesterol reductase activity when it was added to the reaction mixture that contained the radiolabeled substrate.