A bacterial strain (Chol-1S(T)) that is able to oxidize cholesterol to CO2 and reduce nitrate to dinitrogen was enriched and isolated from an upflow sludge bed (USB) anoxic reactor that treats sanitary landfill leachate from the city of Montevideo, Uruguay. Cells of strain Chol-1S(T) were gram-negative, rod-shaped to slightly curved, measured 0.5-0.6 x 1.0-1.3 microm and were motile by a single polar flagellum. Strain Chol-1S(T) grew optimally at 30-32 degrees C and pH 7.0, with a doubling time of 44-46 h when cholesterol was used as the sole carbon and energy source. The metabolism of strain Chol-1S(T) was strictly respiratory, with oxygen or nitrate as the terminal electron acceptor. The presence of ubiquinone Q-8 as the sole respiratory lipoquinone indicated that strain Chol-1S(T) belonged to the beta-subclass of the Proteobacteria. Phosphatidylethanolamine was the predominant polar lipid and the G + C content of the DNA was 65.3 mol%. The fatty acid profile of strain Chol-1S(T), cultivated under denitrifying conditions by using a defined mineral medium supplemented with cholesterol, was characterized by the following major components: summed feature 4 (C16:1 omega7c and/or iso C15:0 2-OH), C16:0, C18:1 omega7c and hydroxy acid C10:0 3-OH. Minor components included C10:0, C11:0, C12:0, C14:0, C15:0, C19:0, C19:0 10-methyl and hydroxylated acids C8:0 3-OH and C16:0 3-OH. Analysis of the 16S rDNA sequence showed that strain Chol-1S(T) represents a separate lineage within the Thauera, Azoarcus, Zoogloea and Rhodocyclus assemblage of the beta-Proteobacteria. Strain Chol-1S(T) had highest sequence similarity (96.5%) with strain 72Chol, a denitrifying beta-Proteobacterium. On the basis of polyphasic evidence, strain Chol-1S(T) (=DSM 13999T=ATCC BAA-354T) is proposed as the type strain of Sterolibacterium denitrificans gen. nov., sp. nov.