A thermophilic, anaerobic, spore-forming bacterium (strain JW/AS-Y6^T) was isolated from a mixed sediment- water sample from a hot spring (Calcite Spring area) at Yellowstone National Park. The vegetative cells of this organism were straight rods, 0.4 to 0.6 by 3.0 to 6.5 µm. Cells occurred singly and exhibited a slight tumbling motility. They formed round refractile endospores in terminal swollen sporangia. Cells stained gram positive. The temperature range for growth at pH 6.8 was 43 to 65°C, with optimum growth at 58°C. The range for growth at 60°C (pH^60C; with the pH meter calibrated at 60°C) was 5.9 to 7.8, with an optimum pH^60C of 6.3 to 6.5. The substrates utilized included glycerol, glucose, fructose, mannose, galactose, xylose, lactate, glycerate, pyruvate, and yeast extract. In the presence of CO₂, acetate was the only organic product from glycerol and carbohydrate fermentation. No H₂ was produced during growth. The strain was not able to grow chemolithotrophically at the expense of H₂-CO₂; however, suspensions of cells in the exponential growth phase consumed H₂. The bacterium reduced fumarate to succinate and thiosulfate to elemental sulfur. Growth was inhibited by ampicillin, chloramphenicol, erythromycin, rifampin, and tetracycline, but not by streptomycin. The G1C content of the DNA was 54.5 mol% (as determined by high-performance liquid chromatography). The 16S ribosomal DNA sequence analysis placed the isolate in the Gram type-positive Bacillus-Clostridium subphylum. On the basis of physiological properties and phylogenetic analysis we propose that the isolated strain constitutes a new species, Moorella glycerini; the type strain is JW/AS-Y6 (5 DSM 11254 5 ATCC 700316).