Minimal requirements of amino acids and vitamins were determined in chemically defined medium for five strains of Clostridium difficile. Cysteine, isoleucine, leucine, proline, tryptophan and valine were essential amino acids for growth of C. difficile. Arginine, glycine, histidine, methionine and threonine enhanced growth. Biotin, pantothenate and pyridoxine were essential vitamins. A defined medium containing the minimal requirements of amino acids and vitamins produced a rapid and heavy growth which was comparable to that in modified brain heart infusion, a complex medium. Adenine was able to substitute for glycine and threonine, suggesting that the two amino acids may be utilized as precursors of purine nucleotides. The defined medium developed here will assist physiological and biochemical studies on C. difficile.