Megasphaera elsdenii B159 (formerly Peptostreptococcus elsdenii; Rogosa 1971) is able to grow in a chemically defined medium containing minerals, NH4+ as the sole nitrogen source, sulfate as the source of sulfur, acetate and glucose as the carbon and energy sources, and the vitamins biotin, pyridoxine, and calcium pantothenate. When lactate serves as the carbon source, with cysteine present as the reducing agent, acetate is not required. Valine and threonine appeared to be required although direct inoculation of unwashed cells into media lacking these amino acids permitted growth in their absence after an extended incubation period. Sulfur sources utilized included sulfate, sulfide, thiosulfate, cysteine, and glutathionine; methionine was utilized less readily. When glucose was autoclaved in the medium, the generation time of M. elsdenii was 170--200 min, but increased to more than 400 min when glucose was autoclaved separately and added to the cooled sterile medium. When fructose, which had been sterilized separately, was utilized as the carbon and energy source, the generation time was 200 min.