A novel cholesterol-reducing bacterium, Eubacterium coprostanoligenes ATCC 51222, was characterized and tested for its hypocholesterolemic effects in animal models. The bacterium did not require cholesterol for growth and synthesized cholesterol reductase constitutively. Lecithin was required for growth but excess lecithin in media did not improve bacterial growth and coprostanol production. Addition of pyruvate, lactose, and most reducing agents in media increased bacterial growth and coprostanol production. The bacterium was sensitive to most antibiotics but had strong resistance to tetracycline. A resting cell assay was established to evaluate the cholesterol reductase activity. E. coprostanoligenes reduced cholesterol through an indirect pathway. Intermediates were detected in resting cell assays even though no accumulation of intermediates was observed in actively growing cultures. E. coprostanoligenes seemed to have a broad specificity for substrates. In resting cell assays, NADH or FAD increased cholesterol reductase activity, whereas EDTA inhibited the activity, and HgCl2 abolished the cholesterol reductase activity. Attempts to achieve active cell-free extracts were not successful because a great loss of activity occurred after the disruption of bacterial cells. The genomic expression library of E. coprostanoligenes was constructed in an expression vector, bacteriophage [lambda] vector [lambda]gt11. Much effort was made to develop a screening technique for cholesterol reductase-containing clones. Effects of orally administered E. coprostanoligenes on plasma cholesterol concentrations were studied in rabbits, laying hens, and germ-free and conventional mice. Oral administration of E. coprostanoligenes caused a significant hypocholesterolemic effect in dietary-induced hypercholesterolemic rabbits, and this effect can be explained by increased conversion of cholesterol to coprostanol in the intestine. In normocholesterolemic laying hens, the bacteria colonized in the intestine but did not affect plasma cholesterol concentrations. E. coprostanoligenes did not colonize in the intestine of germ-free and conventional mice after bacterial feeding. In summary, the study has shown that E. coprostanoligenes reduced cholesterol to coprostanol in vitro and in vivo. The results suggest a potential application of E. coprostanoligenes in humans to decrease plasma cholesterol concentration naturally in hypercholesterolemic individuals.