We characterized two isolates of cholesterol-reducing Eubacterium by conducting conventional biochemical tests and by testing various sterols and glycerolipids as potential growth factors. In media containing cholesterol and plasmenylethanolamine, the tests for nitrate reduction, indole production, and gelatin and starch hydrolyses were negative, and no acid was produced from any of 22 carbohydrates. Both isolates hydrolyzed esculin to esculetin, indicating ,B-glycosidase activity. In addition to plasmenylethanolamine, five other lipids which contain an alkenyl ether residue supported growth of Eubacterium strain 403 in a lecithin-cholesterol base medium. Of six steroids tested, cholesterol, cholest-4en-3-one, cholest-4-en-3,8-ol (allocholesterol), and androst-5-en-3,B-ol-17-one supported growth of Eubacterium strain 403. All four steroids were reduced to the cultures in base medium without carbohydrate. Nitrate reduction, indole production, and starch, gelatin, and esculin hydrolyses were tested as indicated by Holdeman and Moore (8).